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.     

Focal degeneration of astrocytes in amyotrophic lateral sclerosis

Astrocytes emerge as key players in motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). Whether astrocytes cause direct damage by releasing toxic factors or contribute indirectly through the loss of physiological functions is unclear. Here we identify in the hSOD1(G93A) transgenic mouse model of ALS a degenerative process of the astrocytes, restricted to those directly surrounding spinal motor neurons. This phenomenon manifests with an early onset and becomes significant concomitant with the loss of motor cells and the appearance of clinical symptoms. Contrary to wild-type astrocytes, mutant hSOD1-expressing astrocytes are highly vulnerable to glutamate and undergo cell death mediated by the metabotropic type-5 receptor (mGluR5). Blocking mGluR5 in vivo slows down astrocytic degeneration, delays the onset of the disease and slightly extends survival in hSOD1(G93A) transgenic mice. We propose that excitotoxicity in ALS affects both motor neurons and astrocytes, favouring their local interactive degeneration. This new mechanistic hypothesis has implications for therapeutic interventions.     

Dissemination of extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow

Toxoplasma gondii is an intracellular parasite. It has been thought that T. gondii can disseminate throughout the body by circulation of tachyzoite-infected leukocytes (intracellular parasite) in the blood flow. However, a small number of parasites exist as free extracellular tachyzoites in the blood flow (extracellular parasite). It is still controversial whether the extracellular parasites in the blood flow disseminate into the peripheral tissues. In this study, we evaluated the dissemination efficiency of the extracellular and intracellular parasites in the blood flow using GFP-expressing transgenic parasite (PLK/GFP) and DsRed Express-expressing transgenic parasite (PLK/RED). When PLK/GFP and PLK/RED tachyzoites were injected, as intracellular and extracellular forms respectively, at the same time into the tail vein of a mouse, many disseminated green fluorescent PLK/GFP tachyzoites were observed in the lung, the spleen, the liver and the brain. However, only a few red fluorescent PLK/RED tachyzoites were detected in these organs. When PLK/GFP and PLK/RED tachyzoites were injected in the opposite manner, that is, as extracellular and intracellular forms respectively, the majority of tachyzoites in these tissues were PLK/RED tachyzoites. Collectively, these results indicate that intracellular tachyzoites mainly disseminate throughout the body and that extracellular tachyzoites hardly contribute to parasite dissemination.     

6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.