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LPS is recognized by TLR4 and radioprotective 105 kDa in B cells. Susceptibility to LPS in murine B cells is most closely linked to the locus containing the TLR4 gene. However, the molecular mechanism underlying genetic control of LPS sensitivity by this locus has not been fully elucidated. In this study, we revealed that C57BL/6 (B6) B cells respond to mAb-induced, TLR4-specific signals stronger than BALB/c (BALB) B cells, as assessed by proliferation and upregulation of CD69 and CD86. In contrast, BALB B cells were not hyporesponsive to agonistic anti-radioprotective 105 kDa mAb or the TLR9 agonist CpG. Although the level of TLR4 mRNA in BALB B cells was comparable with that in B6 B cells, surface TLR4 expression in BALB B cells was lower than that in B6 B cells. This lower surface expression of BALB TLR4 was also observed when HEK293 and Ba/F3 cells were transfected with a BALB TLR4 expression construct. We identified a V254I mutation as the responsible single nucleotide polymorphism for lower surface expression of BALB TLR4. Furthermore, cotransfection of myeloid differentiation factor-2 increased BALB TLR4 expression, although it was still lower than B6 TLR4 expression. In concordance with reduced expression, Ba/F3 cells transfected with BALB TLR4 and myeloid differentiation factor-2 were hyporesponsive compared with those with B6 TLR4, as assessed by LPS-induced NF-kB activation. In conclusion, we revealed that LPS sensitivity is genetically controlled by the level of surface TLR4 expression on B cells. A V254I mutation accounts for the LPS hyporesponsive phenotype of BALB B cells. [ABSTRACT FROM AUTHOR]

Recognition of LPS by the toll-like receptor 4 (TLR4)/MD-2 complex is a trigger of innate immune defense against bacterial invasion. However, excessive immune activation by this receptor complex causes septic shock and autoimmunity. Manipulation of TLR4 signaling represents a potential therapy that would avoid the detrimental consequences of unnecessary immune responses. In this study, we established two novel mAbs that inhibit LPS-induced human TLR4 activation. HT52 and HT4 mAbs inhibited LPS-induced nuclear factor-κB activation in TLR4/MD-2-expressing Ba/F3-transfected cells and cytokine production and up-regulation of CD86 in the human cell line U373 and PBMCs. These inhibitory activities were stronger than that of HTA125 mAb, which we previously reported. Immunofluorescent and biochemical studies using TLR4 deletion mutants revealed that HT52 and HT4 recognized spatially distinct regions on TLR4 irrespective of MD-2 association. The HT52 and HTA125 epitopes were localized within aa 50–190, while the HT4 epitope was formed only by the full length of TLR4. In addition, we demonstrated that HT52 and HT4 failed to compete with LPS for binding to TLR4/MD-2 but inhibited LPS-induced TLR4 internalization. Inhibitory activities were not due to the interaction with the Fcγ receptor CD32. Our finding that binding of mAbs to at least two distinct regions on TLR4 inhibits LPS-dependent activation provides a novel method for manipulating TLR4 activation and also a rationale for designing drugs targeted to TLR4. [ABSTRACT FROM AUTHOR]

The baculovirus–insect cell expression system is in widespread use for expressing post-translationally modified proteins. As a result, it is potentially applicable for the production of glycoproteins for therapeutic and diagnostic purposes. For practical use, however, remodeling of the biosynthetic pathway of host-cell N-glycosylation is required because insect cells produce paucimannosidic glycoforms, which are different from the typical mammalian glycoform, due to trimming of the non-reducing terminal β1,2-GlcNAc residue of the core structure by a specific β-N-acetylglucosaminidase. In order to establish a cell line which could be used as a host for the baculovirus-based production of glycoproteins with mammalian-type N-glycosylation, we prepared and characterized Spodoptera frugiperda Sf21 cells that had been transfected with the rat cDNA for β1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting GlcNAc. As evidenced by structural analyses of N-glycans prepared from whole cells and the expressed recombinant glycoproteins, the introduction of GnT-III led to the production of bisected hybrid-type N-glycans in which the β1,2-GlcNAc residue at the α1,3-mannosyl branch is completely retained and which has the potential to be present in mammalian cells. These results and other related findings suggest that bisected oligosaccharides are highly resistant to β-N-acetylglucosaminidase activity of the S. frugiperda fused lobes gene product, or other related enzymes, which was confirmed in Sf21 cells. Our present study demonstrates that GnT-III transfection has the potential to be an effective approach in humanizing the N-glycosylation of lepidopteran insect cells, thereby providing a possible preliminary step for the generation of complex-type glycoforms if the presence of a bisecting GlcNAc can be tolerated. [ABSTRACT FROM PUBLISHER]

FUT8, a eukaryotic α1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-β-l-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally similar to that of NodZ, a bacterial α1,6-fucosyltransferase, which acts on a chitooligosaccharide in the synthesis of Nod factor. While the substrate specificities for the nucleotide sugar and the N-glycan have been determined, it is not known whether FUT8 is able to fucosylate other sugar chains such as chitooligosaccharides. The present study was conducted to investigate the action of FUT8 on chitooligosaccharides that are not generally thought to be a substrate in mammals, and the results indicate that FUT8 is able to fucosylate such structures in a manner comparable to NodZ. Surprisingly, structural analyses of the fucosylated products by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance indicated that FUT8 does not utilize the reducing terminal GlcNAc for fucose transfer but shows a preference for the third GlcNAc residue from the nonreducing terminus of the acceptor. These findings suggest that FUT8 catalyzes the fucosylation of chitooligosaccharide analogous to NodZ, but that a nonreducing terminal chitotriose structure is required for the reaction. The substrate recognition by which FUT8 selects the position to fucosylate might be distinct from that for NodZ and could be due to structural factor requirements which are inherent in FUT8. [ABSTRACT FROM PUBLISHER]

Carbonyl compounds, which are naturally produced and augmented under oxidative stress, have deleterious effects on the reproductive system. The aldo-keto reductase (AKR) family of enzymes catalyze the reductive detoxification of various carbonyl compounds in an NADPH-dependent manner. To elucidate involvement of AKR in detoxification of endogenously produced carbonyls in the male reproductive system, we investigated the differential expression and tissue localization of aldehyde reductase (ALR) and protein adducts produced by reaction with lipid peroxidation products. A strong immunoreactivity to an anti-ALR antibody was observed in the epithelia of the epididymis, vas deferens, seminal vesicle, and prostate gland. Virtually the same cells were stained with a monoclonal antibody (mAb) 5F6, raised against an acrolein-modified protein. In the testis, however, mAb5F6 specifically stained the nuclei of somatic cells and less differentiated spermatogenic cells. While acrolein inactivated glutathione reductase, an enzyme involved in recycling oxidized glutathione, AKR activity was affected at the high concentration only. The colocalization of lipid peroxidation products and AKR in the epithelia of the male genital tract indicates that these tissues are exposed to oxidative stress and possess a protective system coordinately. [ABSTRACT FROM AUTHOR]

Histochemical examination of mouse tissues showed nuclear staining of extracellular superoxide dismutase (EC-SOD), and the nuclear translocation of EC-SOD was also confirmed in cultured cells that had been transfected with its gene, as shown by immunohistochemistry and Western blot analysis. The EC-SOD which was secreted into the medium was incorporated into 3T3-L1 cells and a significant fraction of the material taken up was localized in the nucleus. Site-directed mutagenesis indicated that the heparin-binding domain of EC-SOD functions as the nuclear localization signal. These results suggest that the mechanism of the nuclear transport of EC-SOD involves a series of N-terminal signal peptide- and C-terminal heparin-binding domain-dependent processes of secretion, re-uptake and the subsequent nuclear translocation. The findings herein provide support for the view that the role of EC-SOD is to protect the genome DNA from damage by reactive oxygen species and/or the transcriptional regulation of redox-sensitive gene expression. [Copyright &y& Elsevier]

It is well known that numerous cancer-related changes occur in glycans that are attached to glycoproteins, glycolipids and proteoglycans on the cell surface and these changes in structure and the expression of the glycans are largely regulated by glycosyl-transferases, glycosidases, nucleotide sugars and their related genes. Such structural changes in glycans on cell surface proteins may accelerate the progression, invasion and metastasis of cancer cells. Among the over 200 known glycosyltransferases and related genes, β 1,6 N-acetylglucosaminyltransferase V (GnT-V) (the MGAT5 gene) and α 1,6 fucosyltransferase (FUT8) (the FUT8 gene) are representative enzymes in this respect because changes in glycans caused by these genes appear to be related to cancer metastasis and invasion in vitro as well as in vivo, and a number of reports on these genes in related to epithelial-mesenchymal transition (EMT) have also appeared. Another enzyme, one of the N-glycan branching enzymes, β1,4 N-acetylglucosaminyltransferase III (GnT-III) (the MGAT3 gene) has been reported to suppress EMT. However, there are intermediate states between EMT and mesenchymal-epithelial transition (MET) and some of these genes have been implicated in both EMT and MET and are also probably in an intermediate state. Therefore, it would be difficult to clearly define which specific glycosyltransferase is involved in EMT or MET or an intermediate state. The significance of EMT and N-glycan branching glycosyltransferases needs to be reconsidered and the inhibition of their corresponding genes would also be desirable in therapeutics. This review mainly focuses on GnT-III, GnT-V and FUT8, major players as N-glycan branching enzymes in cancer in relation to EMT programs, and also discusses the catalytic mechanisms of GnT-V and FUT8 whose crystal structures have now been obtained. • GnT-III, GnT-V and FUT8, major players as N-glycan branching enzymes are implicated in various diseases including cancer. • These enzymes act as biomarkers for the EMT (epithelial-mesenchymal transition) programs and modify various cell surface proteins. • E-cadherin, integrins and related molecules are target proteins of these enzymes and are highly implicated in EMT programs. • TGF-b and core EMT transcriptional factors play key roles in the programs. [ABSTRACT FROM AUTHOR]

Previous structural analyses showed that human α1,6-fucosyltransferase, FUT8 contains a catalytic domain along with two additional domains, N-terminal α-helical domain and C-terminal Src homology 3 domain, but these domains are unique to FUT8 among glycosyltransferases. The role that these domains play in formation of the active form of FUT8 has not been investigated. This study reports on attempts to determine the involvement of these domains in the functions of FUT8. Based on molecular modeling, the domain mutants were constructed by truncation and site-directed mutagenesis, and were heterologously expressed in Sf21 or COS-1 cells. The mutants were analyzed by SDS-PAGE and assayed for enzymatic activity. In vivo cross-linking experiments by introducing disulfide bonds were also carried out to examine the orientation of the domains in the molecular assembly. Mutagenesis and molecular modeling findings suggest that human FUT8 potentially forms homodimer in vivo via intermolecular hydrophobic interactions involving α-helical domains. Truncation or site-directed mutagenesis findings indicated that α-helical and SH3 domains are all required for enzymatic activity. In addition, in vivo cross-linking experiments clearly indicated that the SH3 domain located in close proximity to the α-helical domain in an intermolecular manner. α-Helical and SH3 domains are required for a fully active enzyme, and are also involved in homophilic dimerization, which probably results in the formation of the active form of human FUT8. α-Helical and SH3 domains, which are not commonly found in glycosyltransferases, play roles in the formation of the functional quaternary structure of human FUT8. • Human α1,6-fucosyltransferase, FUT8 was proposed to be a homodimer. • α-Helical and SH3 domains are required for the enzymatic activity of FUT8. • The hydrophobic interaction of α-helical domain is involved in the dimerization. • The spatial orientation of SH3 domain is involved in the dimerization of FUT8. [ABSTRACT FROM AUTHOR]

The emergence of nontrivial topological order in condensed matter has been attracting a great deal of attention owing to its promising technological applications in novel functional nanodevices. In ferroelectrics, the realization of polar topological order at an ultimately small scale is extremely challenging due to the lack of chiral interaction and the critical size of the ferroelectricity. Here, we break through these limitations and demonstrate that the ultimate atomic-scale polar skyrmion and meron (∼2 nm) can be induced by engineering oxygen vacancies on the SrTiO 3 (001) surface based on first-principles calculations. The paraelectric-to-antiferrodistortive phase transition leads to a novel topological transition from skyrmion to meron, indicating phase-topology correlations. We also discuss accumulating and driving polar skyrmions based on the oxygen divacancy model; these results and the recent discovery of defect engineering techniques suggest the possibility of arithmetic operations on topological numbers through the natural self-organization and diffusion features of oxygen vacancies.

L-γ-Glutamyl-L-cysteinyl-glycine is commonly referred to as glutathione (GSH); this ubiquitous thiol plays essential roles in animal life. Conjugation and electron donation to enzymes such as glutathione peroxidase (GPX) are prominent functions of GSH. Cellular glutathione balance is robustly maintained via regulated synthesis, which is catalyzed via the coordination of γ-glutamyl-cysteine synthetase (γ-GCS) and glutathione synthetase, as well as by reductive recycling by glutathione reductase. A prevailing short supply of L-cysteine (Cys) tends to limit glutathione synthesis, which leads to the production of various other γ-glutamyl peptides due to the unique enzymatic properties of γ-GCS. Extracellular degradation of glutathione by γ-glutamyltransferase (GGT) is a dominant source of Cys for some cells. GGT catalyzes the hydrolytic removal of the γ-glutamyl group of glutathione or transfers it to amino acids or to dipeptides outside cells. Such processes depend on an abundance of acceptor substrates. However, the physiological roles of extracellularly preserved γ-glutamyl peptides have long been unclear. The identification of γ-glutamyl peptides, such as glutathione, as allosteric modulators of calcium-sensing receptors (CaSRs) could provide insights into the significance of the preservation of γ-glutamyl peptides. It is conceivable that GGT could generate a new class of intercellular messaging molecules in response to extracellular microenvironments.

A 6-month-old Shiba Inu dog was brought to the Veterinary Medical Teaching Hospital because of a cough, exercise intolerance, and pulmonary edema. The dog had a Levine 2/6 systolic murmur. Transthoracic echocardiography revealed left atrial and ventricular dilatation (left atrium to aortic ratio: 2.8), mitral and tricuspid valve regurgitation, and severe left ventricular myocardial hypokinesia (fractional shortening was 11.8%). Bubble contrast echocardiography did not reveal a congenital shunt; therefore, the dog was clinically diagnosed with early onset dilated cardiomyopathy. From the first visit, the dog was treated with pimobendan, taurine, torasemide, and isosorbide dinitrate. After 435 days, echocardiography revealed that systolic function had not improved. On Day 465, atrial fibrillation was confirmed via electrocardiogram, and treatment with diltiazem hydrochloride was initiated. The dog continued to appear clinically stable thereafter, until it died suddenly 1087 days after the initial visit. A postmortem histopathological examination identified severe enlargement of the left atrial and ventricular chambers as well as attenuated wavy fibers in the ventricular myocardium, which confirmed dilated cardiomyopathy in a juvenile. This is the first report of a juvenile form of dilated cardiomyopathy in a Shiba Inu dog. This case report provides evidence that the extended prognosis of this dog differed from that in previously reported cases of dilated cardiomyopathy in young dogs. Key clinical message: This is the first reported case of a juvenile form of dilated cardiomyopathy in a Shiba Inu dog. This report provides evidence that the prognosis of this dog differed from that in previously reported cases of dilated cardiomyopathy in young dogs., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography-mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.

Background: Although iodinated contrast (IC) agents are commonly used in endovascular aneurysm repair (EVAR), perioperative use in patients with renal dysfunction or IC allergies is avoided. Carbon dioxide (CO 2 )-guided angiography is a promising alternative. We aimed to evaluate short-term and midterm outcomes of EVAR using CO 2 -guided angiography., Methods: Three hundred eighty-one patients who underwent EVAR from January 2012 to September 2016 were retrospectively reviewed and divided into an IC-EVAR group (n = 351) and CO 2 -EVAR group (n = 30). Subjects in the CO 2 -EVAR group had severe renal dysfunction (n = 27) and IC allergy (n = 4). Intraoperative, postoperative, and follow-up variables were compared., Results: Compared with the IC-EVAR group, preoperative serum creatinine level was significantly higher (2.0 vs. 0.92 mg/dL, P < 0.0001) and mean IC dose was significantly lower (18 vs. 55 mL, P < 0.0001) in the CO 2 -EVAR group. The fluoroscopy time, operative time, number of stent grafts placed, and technical success rates of the groups were similar; no type I and/or type III endoleaks were detected on completion angiography. There was no acute kidney injury and one case of intestinal necrosis in the CO 2 -EVAR group, potentially due to cholesterol embolism. Postoperative endoleak, enlargement of aneurysms, survival, freedom from secondary intervention, and renal function change up to 3 months, postoperatively, were similar between the groups., Conclusions: CO 2 -EVAR is technically feasible and exhibits prominent renal protection. However, consideration of the aortic lumen status remains an important challenge., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Oxidative stress plays pivotal roles in the progression of lung adenocarcinoma (LUAD) through cell signaling related closely to cancer growth. We previously reported that peroxiredoxin 4 (PRDX4), a secretory-type antioxidant enzyme, can protect against the development of various diseases, including potential malignancies. Since many patients with early-stage LUAD develop recurrence, even after curative complete resection, we investigated the association of the PRDX4 expression with the clinicopathological features and recurrence/prognosis using post-surgical samples of stage I-LUAD. Methods: The expression of PRDX4 and MIB-1, a widely accepted Ki67 protein, was immunohistochemically analysed in 206 paraffin-embedded tumour specimens of patients with stage I-LUAD. The PRDX4 expression was considered to be weak when less than 25% of the adenocarcinoma cells showed positive staining. Results: A weak PRDX4+ expression demonstrated a significantly close relationship with pathologically poor differentiation, highly invasive characteristics and recurrence. The decrease in PRDX4-positivity potentially induced cell growth in LUAD, which was correlated significantly with a very high MIB-1 labelling index (≥17.3%). Univariate/multivariate analyses revealed that the subjects with both weak PRDX4+ expression and a very high MIB-1 index had significantly worse disease-free survival rates than other subjects. Conclusions: The combination of weak PRDX4 expression and a very high MIB-1 index can predict high proliferating activity and recurrence with a potential poor prognosis, especially in post-operative stage I-LUAD patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Aim: To demonstrate the clinical outcomes of a multicenter experience and to suggest guidelines for choosing a suction method., Methods: This retrospective study at 5 medical centers involved 58 consecutive patients undergoing over-the-scope clips (OTSCs) placement. The overall rates of technical success (TSR), clinical success (CSR), complications, and procedure time were analyzed as major outcomes. Subsequently, 56 patients, excluding two cases that used the Anchor device, were divided into two groups: 14 cases of simple suction (SS-group) and 42 cases using the Twin Grasper (TG-group). Secondary evaluation was performed to clarify the predictors of OTSC success., Results: The TSR, CSR, complication rate, and median procedure time were 89.7%, 84.5%, 1.8%, and 8 (range 1-36) min, respectively, demonstrating good outcomes. However, significant differences were observed between the two groups in terms of the mean procedure time (5.9 min vs 14.1 min). The CSR of the SS- and TG-groups among cases with a maximum defect size ≤ 10 mm and immediate or acute refractory bleeding was 100%, which suggests that SS is a better method than TG in terms of time efficacy. The CSR in the SS-group (78.6%), despite the technical success of the SS method (TSR, 100%), tended to decrease due to delayed leakage compared to that in the TG-group (TSR, CSR; 88.1%), indicating that TG may be desirable for leaks and fistulae with defects of the entire layer., Conclusion: OTSC system is a safe and effective therapeutic option for gastrointestinal defects. Individualized selection of the suction method based on particular clinical conditions may contribute to the improvement of OTSC success., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to report.

Peroxiredoxins (PRDXs) form an enzyme family that exhibits peroxidase activity using electrons from thioredoxin and other donor molecules. As the signaling roles of hydrogen peroxide in response to extracellular stimuli have emerged, the involvement of PRDX in the hydrogen peroxide-mediated signaling has become evident. Among six PRDX members in mammalian cells, PRDX4 uniquely possesses a hydrophobic signal peptide at the amino terminus, and, hence, it undergoes either secretion or retention by the endoplasmic reticulum (ER) lumen. The role of PRDX4 as a sulfoxidase in ER is now attracting much attention regarding the oxidative protein folding of nascent proteins. Contrary to this role in the ER, the functional significance of PRDX4 in the extracellular milieu is virtually unknown despite its implications as a biomarker under pathological conditions in some diseases. Other than its systemically expressed form, a variant form of PRDX4 is transcribed from the upstream promoter/exon 1 of the systemic promoter/exon 1 and is uniquely expressed in sexually matured testes. Circumstantial evidence, together with deduced functions from the systemic form, suggests that there are potential roles for testicular PRDX4 in the reproductive processes such as the regulation of hormonal signals and the oxidative packaging of sperm chromatin. Elucidation of these PRDX4 functions under in vivo situations is expected to show the whole picture of how PRDX4 has evolved in multicellular organisms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Synthesis of optically pure δ-lactones by diastereomeric resolution was investigated. Amino acid derivatives, which can be obtained at a relatively low cost, were used as resolving agents. Six optically pure δ-lactones were efficiently synthesized using Cbz-L-alanine without other expensive resolving agents. Both enantiomers of δ-lactone obtained had over 98% enantiomeric excesses. This diastereomeric resolution is very efficient for the preparation of optically pure δ-lactones.

Neural stem/progenitor cells (NPCs) express a variety of asparagine-linked oligosaccharide chains, called N-glycans, on the cell surface, and mainly produce hybrid-type and complex-type N-glycans. However, the expression profiles and roles of N-acetylglucosaminyltransferase-V (GnT-V), an enzyme that forms β1,6-branched N-glycans, in NPCs remain unknown. In this study, cultured NPCs were prepared from adult or embryo cortex, and were maintained as either proliferating NPCs or differentiated cells in vitro. Analysis using reverse-transcriptase polymerase chain reaction, Western blot and lectin blot revealed that GnT-V and its reaction products were distinctly expressed in proliferating NPCs; moreover expression of GnT-V and its reaction products were markedly diminished in differentiated cells. In brain slices, many GnT-V-positive neurogenic cells were detected throughout the cerebral cortex on embryonic day 13, while only a few doublecortin (Dcx)- and GnT-V-double positive NPCs were detected around the subventricular zone of the lateral ventricle in the adult brain. However, in the mice in which motor function was spontaneously recovered after cryoinjury to the motor cortex, many Dcx- and GnT-V-double positive NPCs were found to have accumulated around the brain lesion of the adult cerebral cortex compared with the mice in which the function did not recover. These results indicate that GnT-V expression is under rigorous control during NPC differentiation. Furthermore, expression of GnT-V and its reaction products in NPCs may be necessary for the functional recovery after brain injury, and could be used as a marker for visualization of NPCs., (Copyright © 2014. Published by Elsevier B.V.)

Mammalian γ-glutamyl transpeptidase (GGT) is expressed most highly in the kidney and serves to recover the constituent amino acids of glutathione in the proximal tubules. Serum GGT is used as a marker for obstructive jaundice and alcoholic liver disease and it has been reported that urinary GGT is a potential marker for bone resorption. The present study investigated the effect of derivatives of vitamin D3 on GGT activity in LLC-PK1 porcine renal tubular epithelial cells in order to analyze the biochemical basis of bone metabolism-associated alterations in GGT activity in the kidney. In the presence of 1α,25-dihydroxyvitamin D3 [1,25(OH)2VD3], GGT activity was observed to be significantly increased in LLC-PK1 cells, with an increase in GGT activity also found in the cell medium. While the stimulatory effect of 1-OH-VD3 was similar to that of 1,25(OH)2VD3, vitamin D3 and 25-OH-VD3 had no effect on GGT activity. The increased GGT activity caused by 1,25(OH)2VD3 in LLC-PK1 cells was the result of long-term stimulation of the cells, in contrast to the GGT-induced increase in alkaline phosphatase, which is more transient. Polymerase chain reaction analysis revealed that the 1,25(OH)2VD3‑induced increase in GGT activity was due to prolonged GGT turnover, rather than increased GGT expression, as no increase in GGT mRNA expression was observed. Thus, it is likely that the expression of GGT is not entirely constitutive in the kidney, but is altered under certain conditions, including under hormonal regulation.

Although core α1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, α1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of α1,6-fucosyltransferase in insects, we cloned the cDNA for the α1,6-fucosyltransferase from Bombyx mori (Bmα1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bmα1,6FucT consists of 1737bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other α1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bmα1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bmα1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of α1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We report a case involving a ruptured acute type B aortic dissection originating from an aberrant right subclavian artery (ARSA). A thoracic stent-graft was deployed in the distal arch close to the origin of the ARSA; the entry site at the origin of the ARSA was embolized with metallic coils. Perfusion of the left subclavian artery was preserved without a surgical bypass by using a chimney graft. This procedure is a feasible and less invasive treatment for high-risk sternotomy patients and is an effective strategy for acute aortic dissections involving an ARSA., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Heregulin signaling is involved in various tumor proliferations and invasions; thus, receptors of heregulin are targets for the cancer therapy. In this study we examined the suppressing effects of extracellular domains of ErbB2, ErbB3, and ErbB4 (soluble ErbB (sErbB)) on heregulin β signaling in human breast cancer cell line MCF7. It was found that sErbB3 suppresses ligand-induced activation of ErbB receptors, PI3K/Akt and Ras/Erk pathways most effectively; sErbB2 scarcely suppresses ligand-induced signaling, and sErbB4 suppresses receptor activation at ∼10% efficiency of sErbB3. It was revealed that sErbB3 does not decrease the effective ligands but decreases the effective receptors. By using small interfering RNA (siRNA) for ErbB receptors, we determined that sErbB3 suppresses the heregulin β signaling by interfering ErbB3-containing heterodimers including ErbB2/ErbB3. By introducing the mutation of N418Q to sErbB3, the signaling-inhibitory effects were increased by 2-3-fold. Moreover, the sErbB3 N418Q mutant enhanced anticancer effects of lapatinib more effectively than the wild type. We also determined the structures of N-glycan on Asn-418. Results suggested that the N-glycan-deleted mutant of sErbB3 suppresses heregulin signaling via ErbB3-containing heterodimers more effectively than the wild type. Thus, we demonstrated that the sErbB3 N418Q mutant is a potent inhibitor for heregulin β signaling.

Introduction: We report a rare case of invasive micropapillary carcinoma in the male breast., Presentation of Case: A 63-year-old man was referred to our hospital for investigation of a left breast tumor, which could be palpated in the upper lateral quadrant of the left nipple-areola complex. The tumor invaded the areola skin. Ultrasonography showed a 14.8×15.0×12.4mm low echoic mass, with an irregular lobulated border. Core needle biopsy indicated invasive ductal carcinoma, but the subtype could not be accurately determined. Mastectomy with axillary lymph node dissection was performed. Pathological examination indicated invasive micropapillary carcinoma, no lymph node metastasis, and a nuclear grade of 2. Immunohistochemical examination showed positive staining for estrogen and progesterone receptors, but negative staining for HER2. The Ki67 index was 5%. Tamoxifen was administered, and recurrence has not been noted for 1 year., Discussion: Women's IMPC generally shows a high HER2 positivity rate. However, HER2 positivity was noted in only 1 male patient with IMPC (14%) according to our literature review. Furthermore, in all cases of the mixed type that were reviewed, IMPC was associated with papillotubular carcinoma. These findings may be specific to IMPC in male patients., Conclusion: IMPC is associated with a high rate of lymph node metastasis or recurrence and advanced vessel invasion, aggressive adjuvant chemotherapy following surgical resection should be selected for patients with IMPC., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

N-Glycans with an α-fucose unit linked to the 6-position of the innermost GlcNAc are widely distributed among the animal kingdom, from worms and insects to human. This α1,6-linked fucosyl residue, frequently referred to as a core fucose, is formed via the action of an α1,6-fucosyltransferase, the mammalian ortholog which is systematically called FUT8. In mammals, it is well known that the extent of core-fucosylation in cellular and secreted glycoproteins varies, e.g., according to differentiation and carcinogenesis of the cells. This chapter describes a method for the sensitive and quantitative assay of FUT8 activity using a fluorescence-labeled oligosaccharyl asparagine derivative as the glycosyl acceptor substrate.

Peroxiredoxin (Prx)-4, a secretable endoplasmic reticulum (ER)-resident isoform of the mammalian Prx family, functions as a thioredoxin-dependent peroxidase. It is acknowledged that Prx-4 plays a role in the detoxification of hydrogen peroxide, and potentially other peroxides, which may be generated during the oxidative folding of proteins and oxidative stress in the ER. The present study was undertaken in order to specifically quantify the tissue levels of Prx-4. To accomplish this, an enzyme-linked immunosorbent assay was developed using a specific polyclonal antibody produced by immunizing a rabbit with native recombinant rat Prx-4 protein. The assay was used to detect Prx-4 in the range of 0.1 and 10 ng/ml, and to investigate tissue distribution in rats. Using this immunoassay, we found that the serum levels of Prx-4 were substantially lower in asymptomatic Long-Evans Cinnamon rats, a rat model of Wilson's disease, compared to normal rats. In addition, the treatment of rat hepatoma cells with N-acetylcysteine led to a significant increase in the release of Prx-4 protein into the medium; thus, it appears likely that the secretion of Prx-4 is associated with the redox state within cells. These results suggest that serum Prx-4 has potential for use as a biomarker for hepatic oxidative stress.

Background: Surgical ventricular restoration (SVR) is considered as an effective surgical procedure for patients with ischemic myocardiopathy( ICM). However" surgical treatment for ischemic heart failure (STICH)" trial concluded that adding SVR to coronary artery bypass grafting (CABG) did not relieve symptoms and failed to lower death rate or cardiac rehospitalization as compared with CABG alone., Aim: The aim of this study was to investigate the efficacy of CABG with SVR for ICM., Methods and Results: We retrospectively studied 24 patients who had undergone CABG with or with out SVR for ICM from October 1992 to June 2008. In CABG with SVR group, cardiac symptoms were relieved and the left ventricular end-systolic volume index (LVESVI) was reduced from the baseline significantly. However cardiac symptoms were relieved only in CABG-S [left ventricular end-diastolic dimension (LVDd)<60 mm] group, and not in CABG-L (LVDd≥60 mm) group. LVESVI was not reduced in CABG without SVR group., Conclusion: SVR contributed to relieving the symptoms, and improving the left ventricular function and the long-term survival of patients with especially dilated ICM, which could not be achieved by CABG alone.

Eukaryotic typical 2-Cys type peroxiredoxin (Prx) is inactivated by hyperoxidation of the peroxidatic cysteine to a sulphinic acid in a catalytic cycle-dependent manner. This inactivation process has been well documented for cytosolic isoforms of Prx. However, such a hyperoxidative inactivation has not fully been investigated in Prx-4, a secretable endoplasmic reticulum-resident isoform, in spite of being a typical 2-Cys type, and details of this process are reported herein. As has been observed in many peroxiredoxins, the peroxidase activity of Prx-4 was almost completely inhibited in the reaction with t-butyl hydroperoxide. On the other hand, when H(2)O(2) was used as the substrate, the peroxidase activity significantly remained after oxidative damage. In spite of these different consequences, mass spectrometric analyses indicated that both reactions resulted in the same oxidative damage, i.e. sulphinic acid formation at the peroxidatic cysteine, suggesting that another cysteine in the active site confers the peroxidase activity. As suggested by the analyses using cysteine-substituted mutants sulphinic acid formation at the peroxidatic cysteine may play a role in the development of the possible alternative mechanism, thereby sustaining the peroxidase activity that prefers H(2)O(2).

Marfan syndrome is an inherited connective tissue disorder with ocular, skeletal and cardiovascular systems and often causes acute aortic dissection. Interestingly, there have been several reports of familial thoracic aortic dissection in patients with autosomal dominant diseases without Marfan syndrome. Variation of the transforming growth factor-beta receptor (TGFBR) gene is reported to be one of the causes. We report a case of a familial aortic dissection not associated with Marfan syndrome, with mutation of TGFBR type 1. Hereditary aortic dissection of the non-Marfan syndrome that does not have clinical manifestations is not uncommon. Thus, the existence of familial aortic aneurysm should be in mind in diagnosis and treatment.

Glycoconjugates play pivotal and crucial roles in various biological processes, and it is thus believed that abnormal structural changes in the sugar chains of glycoconjugates are closely associated with the pathophysiology and clinical implications of many diseases. Many studies have identified and characterized the genes for glycosyltransferases and other related enzymes that are involved in the biosynthesis of sugar chains. Functional analyses of sugar chains using genetically engineered cells and organisms have clarified the biochemical bases for both the assembly and disease-related structural alteration of sugar chains. The findings obtained to date have been successfully applied to improve the diagnostic value of oligosaccharide biomarkers for diseases and also contributed to the establishment and/or development of therapeutic glycoproteins such as antibody-based drugs and some biofactors. In this article, the contribution and application of glycoscience to medicine, particularly clinical chemistry, will be described.

Peroxiredoxins (Prxs), a family of thioredoxin-dependent peroxidases, are highly conserved in many organisms and function in detoxifying reactive oxygen species as well as other cellular processes. Six members of the Prx family are known in mammals, i.e., Prx-1 through -6. Among these proteins, only Prx-4 appears to contain a signal peptide that serves for localization in the endoplasmic reticulum, membrane translocation and secretion into the extracellular space, as demonstrated in a previous study using a baculovirus-insect cell system. The present study was conducted to determine whether the signal peptide-truncated mutant of rat Prx-4 is expressed as an enzymatically active form and is produced in large amounts. Two N-terminally truncated mutants were prepared by deletion of only the signal peptide and the larger region encompassing both the signal and the unique extension to Prx-4. These mutants were successfully produced within Spodoptera frugiperda 21 cells by infection with the recombinant baculoviruses, rather than by extracellular secretion. Both mutants were efficiently purified to homogeneity by two column chromatography steps. Biochemical characterization of the purified proteins showed that the truncated enzymes are enzymatically active and form an oligomeric structure, as reported for the mammalian Prx family. The findings also suggest that the unique extension plays a role in the regulation of non-covalent oligomerization. More than 4 mg of the purified proteins can be obtained from cells grown in monolayer cultures in twenty 75 cm(2) tissue culture flasks. The procedures described in this study permit recombinant Prx-4 to be prepared more efficiently and easily for purposes of crystallization and antibody preparation., (Copyright 2010. Published by Elsevier Inc.)

Alpha1,6-fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of alpha1,6 core fucose to the innermost N-acetylglucosamine residue of the N-glycan, has been implicated in the development, immune system, and tumorigenesis. We found that alpha1,6-fucosyltransferase and E-cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E-cadherin appeared as well as normal sized E-cadherin in cancer samples. To investigate the correlation between alpha1,6-fucosyltransferase and E-cadherin expression, we introduced alpha1,6-fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E-cadherin was significantly increased in alpha1,6-fucosyltransferase-transfected WiDr cells in dense culture, which resulted in an enhancement in cell-cell adhesion. The transfection of mutated alpha1,6-fucosyltransferase with no enzymatic activity had no effect on E-cadherin expression, indicating that core fucosylation is involved in the phenomena. In alpha1,6-fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E-cadherin and E-cadherin dependent cell-cell adhesion was decreased. The introduction of alpha1,6-fucosyltransferase into kidney epithelial cells from alpha1,6-fucosyltransferase(-/-) mice restored the expression of E-cadherin and E-cadherin-dependent cell-cell adhesion. Based on the results of lectin blotting, peptide N-glycosidase F treatment, and pulse-chase studies, it was demonstrated that the low molecular weight population of E-cadherin contains peptide N-glycosidase F insensitive sugar chains, and the turnover rate of E-cadherin was reduced in alpha1,6-Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E-cadherin. These results suggest a possible role of core fucosylation in the regulation of cell-cell adhesion in cancer.

Prx (peroxiredoxin) is a multifunctional redox protein with thioredoxin-dependent peroxidase activity. Prx4 is present as a secretory protein in most tissues, whereas in sexually mature testes it is anchored in the ER (endoplasmic reticulum) membrane of spermatogenic cells via an uncleaved N-terminal hydrophobic peptide. We generated a Prx4 knockout mouse to investigate the function of Prx4 in vivo. Prx4(-/y) mice lacking Prx4 expression in all cells were obtained by mating Prx4(flox/+) female mice with Cre-transgenic male mice that ubiquitously expressed Cre recombinase. The resulting Prx4(-/y) male mice were fertile, and most organs were nearly normal in size, except for testicular atrophy. The number of deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive spermatogenic cells was higher in Prx4(-/y) mice than in Prx4(+/y) mice and increased remarkably in response to warming the lower abdomen at 43 degrees C for 15 min. Cells reactive to antibodies against 4-hydroxynonenal and 8-hydroxyguanine were high in the Prx4(-/y) mice and concomitant with elevated oxidation of lipid and protein thiols. The cauda epididymis of Prx4(-/y) mice contained round spermatocytes, which were not found in Prx4(+/y) mice, and displayed oligozoospermia. However, mature spermatozoa from the epididymis of Prx4(-/y) mice exhibited normal fertilization In vitro. Taken together, these results indicate that spermatogenic cells lacking Prx4 are more susceptible to cell death via oxidative damage than their wild-type counterparts. Our results suggest that the presence of Prx4, most likely the membrane-bound form, is important for spermatogenesis, but not an absolute requisite.

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of the bisecting GlcNAc and plays a regulatory role in the biosynthesis of the N-linked oligosaccharide. In this study, we examined whether the glycosyl transfer catalyzed by GnT-III is reversible, and, in addition, investigated the equilibrium of the GnT-III-catalyzed reaction. Incubation of the agalactosyl-bisected biantennary oligosaccharide with GnT-III in the presence of the sufficiently high concentration of uridine diphosphate (UDP) resulted in conversion of the bisected oligosaccharide into the nonbisected one. This reaction was accompanied by the stoichiometric formation of UDP-GlcNAc, which appeared to result from the transfer of GlcNAc from the oligosaccharide to UDP. Thus, these results indicate that GnT-III is capable of perceivably catalyzing the reverse reaction in vitro, as found in some glycosyltransferases. When the equilibrium of the reaction was kinetically analyzed, it was found that the state of the equilibrium is greatly displaced toward the formation of the bisecting GlcNAc. In terms of free energy change, as estimated, the reaction by GnT-III can be comparable to the hydrolysis of ATP. Although GnT-III catalyzes bidirectional transfer of GlcNAc between the oligosaccharide and UDP, the removal of the bisecting GlcNAc is unlikely in vivo, due to the displacement of the equilibrium. It is known that equilibria of certain glycosyltransferase reactions are not biased as greatly as the case of GnT-III, and thus it seems likely that there are a variety of equilibrium states in glycosyltransferase reactions. In living cells, the assembly of oligosaccharides could be regulated by not only rate control but also equilibrium control.

Objective: The right gastroepiploic artery (RGEA) is widely used as an in situ arterial graft for coronary artery bypass grafting (CABG); however, it is impossible to measure an RGEA or check for calcification or stenosis and assess its suitability as a graft before angiography or harvest. We evaluated the accuracy of preoperative three-dimensional computed tomographic angiography (3D-CTA) for assessing the suitability of RGEAs for CABG., Method: We used 4-channel multidetector-row computed tomography with intravenous contrast medium. All the RGEAs had an intraluminar diameter greater than 1.5 mm. RGEAs longer than two-thirds of the greater curvature of stomach, longer than half of the greater curvature, and shorter than half of the greater curvature were defined as large, moderate, and small, respectively., Result: Of the 36 patients examined, 5 (14%) had a small RGEA, 16 (44%) had a moderate RGEA, and 15 (42%) had a large RGEA. We confirmed intraoperatively that two small RGEAs were unsuitable for grafting because they could not reach the posterior descending artery (PDA). The other three small RGEAs were not used. Two of the large and moderate RGEAs with diffuse narrowing and severe calcification were also unsuitable for grafting. This eliminated the need for a laparotomy to harvest the RGEA in five (14%) patients. Intraoperative findings confirmed that all the moderate RGEAs could be anastomosed to the PDA. All the large RGEAs reached the posterolateral artery (PLA), and more than half reached the PLA branching circumflex artery., Conclusion: Preoperative noninvasive evaluation by 3D-CT is effective for assessing the suitability of RGEAs for CABG.

Adenylyl cyclases (ACs) catalyze the synthesis of cAMP in response to extracellular and intracellular signals and are responsible for a wide variety of biological activities including cell growth, differentiation, and metabolism. There are nine, currently known, isoforms of transmembrane ACs, and the primary structure of the catalytic unit and the potential N-glycosylation sites are highly conserved among them. The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to N-glycans. We have been studying the function of GnT-III on signaling molecules. In this study, we report on the effects of a bisecting GlcNAc on AC signaling. We established GnT-III stable expressing cell lines of Neuro-2a mouse neuroblastoma cells and B16 mouse melanoma cells. Forskolin-induced AC activation and downstream signaling, such as the synthesis of cAMP and the phosphorylation of transcriptional factor CRE-binding protein were upregulated in the GnT-III transfectants compared with mock transfectants or a dominant negative mutant of GnT-III-transfected cells. Since endogenous AC expression levels in Neuro-2a and B16 cells were too low to permit the glycosylation status to be examined, AC type III (ACIII) was overexpressed in a stable expression system using Flp-In-293 cells. The N-glycans of ACIII in the GnT-III transfectants were confirmed to be modified by the introduction of a bisecting GlcNAc, and AC activity was found to be significantly up-regulated in the GnT-III transfectants. Thus, the structure of N-glycans of ACIII regulates its enzymatic activity and downstream signaling.

Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction.

Patients with liver cirrhosis are prone to the development of severe complications associated with high mortality rates after major surgery, especially cardiac surgery using cardiopulmonary bypass (CPB). We report the case of a 65-year-old man with acute infective endocarditis and aortic valve perforation, complicated by non-cardiac liver cirrhosis (Child-Pugh class B). After careful preoperative anti-inflammatory and systemic support treatment, we successfully treated infective endocarditis-induced aortic valve perforation by performing aortic valve replacement (AVR).

The core fucosylation (alpha1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated alpha1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8(-/-) mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8(-/-) mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-beta1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8(-/-) mice have a marked dysregulation of TGF-beta1 receptor activation and signaling, as assessed by TGF-beta1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-beta1 receptor defects found in Fut8(-/-) cells can be rescued by reintroducing Fut8 into Fut8(-/-) cells. Furthermore, exogenous TGF-beta1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8(-/-) lung. We propose that the lack of core fucosylation of TGF-beta1 receptors is crucial for a developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.

Subepicardial aneurysm of the left ventricle is a rare complication of myocardial infarction. We report a case of a 75-year-old woman found to have a subepicardial aneurysm 4 months after an acute myocardial infarction. The patient was admitted to our hospital with congestive heart failure, and transthoracic echocardiography and left ventriculography showed an aneurysm on the posterior wall of the left ventricle, 4.6 x 3.5 cm in size. We performed patch repair using a Hemashield graft, with coronary artery bypass grafting. She was discharged on postoperative day 69, and has been well since.

gamma-Glutamyltranspeptidase is a heterodimeric glycoprotein that catalyzes the transpeptidation and hydrolysis of the gamma-glutamyl group of glutathione and related compounds. It is known that the enzyme plays a role in the metabolism of glutathione and in salvaging constituents of glutathione. In the adult animal, high levels of gamma-glutamyltranspeptidase are constitutively expressed in the kidney, intestine, and epididymis. On the other hand, although gamma-glutamyltranspeptidase is up-regulated in the liver during the perinatal stage, its expression is nearly undetectable in the adult. In addition, it has long been observed that the intake of certain xenobiotics, including carcinogens and drugs, induces the hepatic expression of the enzyme. This induction seems to be associated with both transcriptional regulation and the growth of certain types of cells in the injured liver. A number of studies have been carried out to explain the mechanism by which gamma-glutamyltranspeptidase expression is regulated. 5'-Untranslated regions of mRNAs of the enzyme differ in a tissue-specific manner but share a common protein coding region, and the tissue-specific and developmental stage-specific expression, as well as hepatic induction, are conferred by different promoters. As suggested by the capability of enzymatic activity-independent induction of osteoclasts, the expression of gamma-glutamyltranspeptidase may also be involved in various biological processes that are not directly associated with glutathione metabolism. This chapter briefly summarizes studies to date concerning the tissue-specific expression and induction of gamma-glutamyltranspeptidase and transcriptional regulation by the multiple promoter system is discussed.

A novel bone-resorbing factor was cloned using an expression cloning technique, which involved a Xenopus oocyte expression system and an assay for osteoclast formation. A candidate clone was isolated from a BW5147 mouse T-lymphoma cell cDNA library. Sequencing analysis identified the factor as gamma-glutamyltranspeptidase (GGT), which is an enzyme involved in glutathione metabolism. The addition of purified GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. An antibody against GGT inhibited osteoclast formation but not the enzymatic activity. We also demonstrated that an inactive form of GGT, the enzymatic activity of which had been blocked by chemical modification with a specific inhibitor, acivicin, supported osteoclast formation. These results indicate that GGT acts on osteoclast formation independent of its own enzymatic activity. Furthermore, both native GGT and inactive GGT stimulated the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein from bone marrow stromal cells. This report is the first demonstration of a novel biological activity of GGT protein in a manner independent of its enzymatic activity.

Secreted proteins and membrane proteins are frequently post-translationally modified by oligosaccharides. Therefore, many glycoproteins are involved in signal transduction. One example is growth factor receptors, which are membrane proteins that often contain oligosaccharides. The oligosaccharides in those growth factor receptors play crucial roles in receptor functions. An analysis of glycosyltransferase-transfectants revealed that the branching structures of oligosaccharide also serve as important determinants. For example, N-glycans of epidermal growth factor receptor (EGFR) are involved in receptor sorting, ligand binding and dimerization. The addition of a bisecting GlcNAc to N-glycans increases the endocytosis of EGFR. N-glycans of Trk, a high affinity nerve growth factor receptor, also affect its function. Thus, oligosaccharides play an important role in growth factor signaling.

beta 1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is significantly homologous to both snail beta 1,4GlcNAc transferase and beta1,4Gal transferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT-III, for enzymatic activity. The overexpression of Asp323-substituted, catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of 'dominant negative glycosyltransferase', identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.