Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.     

Effects of Aluminum on the Growth of Tea Plant and Activation of Antioxidant System

The origin and distribution of carbonic anhydrase (CA), which could accelerate karst processes, are explored in this paper. The soil samples used in the study were collected from four different kinds of karst ecosystems of Southwest China. The results indicate that CA activity could be detected in surface layer soils, and that CA activity varied obviously among the soils in different karst ecosystems. Of the four kinds of karst site, the mean CA activity of the surface soil in Misuga, Liupanshui, which had the lowest vegetation cover, was the lowest with 0.02 U g ^–1 dry soil. Nongla and Jinfu mountain, where there is abundant vegetable species diversity and dense vegetation, had higher mean CA activity in the surface soil with 3.83 U g ^–1 dry soil and 3.13 U g ^–1 dry soil, respectively. Moreover, there was certain difference in CA activities of soils among different kinds of karst landscape in the same kind of karst ecosystem. On the other hand, the higher CA activity could be detected in the soils in the vicinity of the plant roots, and CA activity in soil exhibited both obvious seasonal and vertical changes. These facts implied that plant roots and soil microorganisms might serve as important sources of CA. Besides, the results indicate that CA activity is found in bacteria, actinomycetes and fungi. Actinomycetes had higher intracellular CA activity, while fungi had higher extracellular CA activity. This study shows that higher microbial CA activities are found in Jinfu Mountain, while lower microbial CA activities are found in Misuga, Liupanshui.     

Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.     

Widespread tissue expression of nepenthesin-like aspartic protease genes in Arabidopsis thaliana.

There are approximately 69 genes encoding aspartyl protease homologues in Arabidopsis thaliana, and most of the gene products constitute a novel subfamily of aspartic proteases. However, their physiological roles are largely unknown. As an initial step to shed light on the roles of these nepenthesin-like aspartic proteases (NAPs), a phylogenetic tree was constructed, which indicated that these proteases are classified into several distinct sub-sub-groups. Based on these results, specific primers were designed for genes selected from several of these groups and their tissue expression was investigated using RT-PCR. The results indicated that these genes are widely expressed in several tissues, such as leaves, stems, seeds and pods, suggesting ubiquitous occurrence and multiple functions of the corresponding proteases in the tissues of A. thaliana.     

Horizontal and vertical distribution and demography of euphausiids in the Ross Sea and its adjacent waters in 2004/2005

The horizontal and vertical distribution and population structure of euphausiids in the Ross Sea and its adjacent waters were investigated during the summers of 2004/2005 using stratified towed samples. Nine species of euphausiids occurred in the survey area. Among them, Euphausia triacantha was dominant in biomass north of the southern boundary of the Antarctic circumpolar current (SB). Thysanoessa spp. was widely distributed north of the continental slope, while E. superba was distributed from the SB to the slope, where it showed the highest biomass. Juvenile E. superba was distributed offshore near the SB and remained at the surface, but gravid females were dominant in the slope and mainly occurred in the middle layers (400–600 m). Adult and juvenile E. crystallorophias were found at 200–300 m in the colder water of the continental shelf. In general, the peak biomass of euphausiids was found in the mid layers of the Ross Sea area. The life span and the number of spawns for major species are also discussed.     

Responses of marine organisms to physical and chemical impacts

Studies on the effects of a variety of exogenous and anthropogenic environmental factors, including endocrine disruptors, heavy metals, UV light, high temperature, and others, on marine organisms have been presented at the 2nd Bilateral Seminar Italy–Japan held in November 2006. Reports were discussed in order to reveal the current situation of marine ecosystems, aiming at evaluation and prediction of environmental risks.

Cloning and expression of three formate oxidase genes from Debaryomyces vanrijiae MH201

Debaryomyces vanrijiae MH201 produces formate oxidase (FOD) at estimated pI values by native isoelectric focusing of 5.1, 5.4, and 5.9. We cloned and expressed three formate oxidase cDNAs, FOD1, FOD2, and FDO3, of the yeast using Escherichia coli. The open reading frames of FOD1, FOD2, and FDO3 were 1,731 bp long, and encoded 576-amino acid polypeptides with molecular masses of 64,142, 63,794, and 63,836 Da respectively. Expression of FOD1, FOD2, and FOD3 resulted in the production of three isozymes, with pI values of 5.1, 5.9, and 5.9 respectively. Co-expression of FOD1 and FOD2 and of FOD1 and FOD3 resulted in the production of additional isozymes with pI values, of 5.4. The three amino acid sequences of FOD1, FOD2, and FOD3 contained a consensus motif of a flavin adenine dinucleotide binding site in their N-terminal parts and a glucose-methanol-choline oxidoreductase signature pattern, suggesting that formate oxidase ought to be classified in the glucose-methanol-choline oxidoreductase family.     

Yeast glycobiology and its application

In this review, I describe the yeast glycans and their biosynthetic pathways, especially in the budding yeast Saccharomyces cerevisiae. The biosynthetic pathway of N-glycan in the endoplasmic reticulum is similar to that of mammalian cells, while the pathway in the Golgi is different from that of mammalian cells, but the biosynthetic pathway of O-glycan, mainly composed of O-oligomannoses, appears to be specific to yeast cells. Yeast systems are useful not only to understand the basic mechanisms of glycan synthesis but also to produce therapeutic proteins with human-type glycans. Protein modification by glycosylphosphatidylinositol is one of the major post-translational modifications in which oligosaccharides are involved. The biosynthetic pathway and the physiological function of glycosylphosphatidylinositol in S. cerevisiae are described in relation to lipid microdomains (also called "lipid rafts"), focusing on the latest findings related to lipid remodeling of GPI-anchored proteins.     

Relationship between pancreatic vesicular monoamine transporter 2 (VMAT2) and insulin expression in human pancreas

Vesicular monoamine transporter 2 (VMAT2) is expressed in pancreatic beta cells and has recently been proposed as a target for measurement of beta cell mass in vivo. We questioned, (1) What proportion of beta cells express VMAT2? (2) Is VMAT2 expressed by other pancreatic endocrine or non-endocrine cells? (3) Is the relationship between VMAT2 and insulin expression disturbed in type 1 (T1DM) or type 2 diabetes (T2DM)? Human pancreas (7 non-diabetics, 5 T2DM, 10 T1DM) was immunostained for insulin, VMAT2 and other pancreatic hormones. Most beta cells expressed VMAT2. VMAT2 expression was not changed by the presence of diabetes. In tail of pancreas VMAT2 immunostaining closely correlated with insulin staining. However, VMAT2 was also expressed in some pancreatic polypeptide (PP) cells. Although VMAT2 was not excluded as a target for beta cell mass measurement, expression of VMAT2 in PP cells predicts residual VMAT2 expression in human pancreas even in the absence of beta cells.