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Cellular translation should be precisely controlled in response to extracellular cues. However, knowledge is limited concerning signal transduction-regulated translation. In the present study, phosphorylation was identified in the 40S small subunit ribosomal protein uS7 (Yjr123w/previously called as Rps5) by Ypk1 and Pkc1, AGC family protein kinases in yeast Saccharomyces cerevisiae. Serine residue 223 (Ser223) of uS7 in the conserved C-terminal region was crucial for this phosphorylation event. S223A mutant uS7 caused severe reduction of small ribosomal subunit production, likely due to compromised interaction with Rio2, resulting in both reduced translation and reduced cellular proliferation. Contrary to optimal culture conditions, heat stressed S223A mutant cells exhibited increased heat resistance and induced heat shock proteins. Taken together, an intracellular signal transduction pathway involving Ypk1/Pkc1 seemed to play an important role in ribosome biogenesis and subsequent cellular translation, utilizing uS7 as a substrate. more...

Sphingolipids are a family of eukaryotic lipids biosynthesized from sphingoid long-chain bases (LCBs). Sphingolipids are an essential class of lipids, as their depletion results in cell death. However, acute LCB supplementation is also toxic; thus, proper cellular LCB levels should be maintained. To characterize the "sphingolipid-signaling intercross," we performed a kinome screening assay in which budding yeast protein kinase-knockout strains were screened for resistance to ISP-1, a potent inhibitor of LCB biosynthesis. Here, one pair of such DIR (deletion-mediated ISP-1 resistance) genes, FPK1 and FPK2, was further characterized. Cellular LCB levels increased in the fpk1/2∆ strain, which was hypersensitive to phytosphingosine (PHS), a major LCB species of yeast cells. Concomitantly, this strain acquired resistance to ISP-1. Fpk1 and Fpk2 were involved in two downstream events; that is, ISP-1 uptake due to aminophospholipid flippase and LCB degradation due to LCB4 expression. RSK3, which belongs to the p90-S6K subfamily, was identified as a functional counterpart of Fpk1/2 in mammalian cells as the RSK3 gene functionally complemented the ISP-1-resistant phenotype of fpk1/2∆ cells. more...

Background Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. Methods Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. Results The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d -glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. Conclusions AKR1A plays a predominant role in the reduction of both d -glucuronic acid and d -glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d -glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. General significance AKR1A is a major enzyme that catalyzes the reduction of d -glucuronic acid and d -glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d -glucuronic acid and saccharate. more...

This study reveals the basis for how temporal phosphoregulation of Orm protein controls sphingolipid production in response to stress. Orm protein phosphorylation is highly responsive to sphingoid bases, and Ypk1 protein kinase transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation., Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55–protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner. more...

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB). more...

The infection of feline thymic lymphoma 3201 cells with a cell culture-adapted Petaluma strain of feline immunodeficiency virus (FIV) led to the establishment of survivor cells designated as 3201-S after a productive infection associated with extensive cell killing. 3201-S cells were free of FIV DNA, and were found to express CXCR4, a coreceptor for infection but not CD134, a primary receptor. When 3201-S cells were reinfected with FIV, viral DNA was transiently detectable for 5 days postinfection, indicating that 3201-S cells cannot support the FIV replicative cycle. Furthermore, comparative studies found that in contrast to SDF-1α-responsive 3201 cells, 3201-S cells did not show a flux of Ca2+ in response to SDF-1α, implying that CXCR4 is not functionally active on 3201-S cells. These results suggest that 3201 cells can be heterogeneous in the phenotype of the CXCR4 expressed, and this heterogeneity may account for the differences in susceptibility to FIV. Determining the mechanism(s) within 3201-S cells that restrict FIV could result in therapeutic strategies against FIV infection. more...

Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, beta-amyloid, is thought to cause much of the pathology of Alzheimer's disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, beta-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of beta-amyloid. Both beta-amyloid 40 and beta-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, alpha-secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional beta-amyloid production. more...

Biotin conjugate of the most potent ligand for mouse CD22 was designed and synthesized. The key synthetic step involved the dual capability of Hanessian reaction for debenzylation and simultaneous conversion of the anomeric p-methoxyphenyl group into the corresponding phenylthioglycoside in one step. Competition enzyme-linked immunosorbent assay (ELISA) for testing the binding affinity of synthetic sialosides was developed based on this biotinylated ligand. more...

CD22/Siglec-2 is a B cell membrane-bound lectin that recognizes glycan ligands containing alpha2,6-linked sialic acid, and negatively regulates signaling through the B cell antigen receptor (BCR). Previous studies demonstrated that synthetic sialosides that bind to CD22 augment BCR signaling by inhibiting CD22-mediated BCR regulation. Here we demonstrate that, after antigen stimulation, CD22 forms a cap together with BCR, and translocates to lipid rafts. Both co-capping of CD22 with BCR and translocation of CD22 to lipid rafts were markedly blocked by a synthetic alpha2,6-linked sialic acid, Neu5Gcalpha2-6GalbetaSE. These results strongly suggest that synthetic glycan ligand excludes CD22 from BCR-containing lipid rafts. Because CD22-mediated signal regulation requires phosphorylation of CD22 by Lyn that localizes in lipid rafts and is activated by BCR, synthetic glycan ligand regulates localization of CD22 crucial for signal regulation. more...

Humans and chimpanzees share99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology. more...

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b 5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG–Des2, m b 5 (the membrane form of cytochrome b 5 ) and bovine erythrocyte membrane. The apparent K m and V max of Des2 for the substrate N -octanoylsphinganine were 35 μM and 40 nmol·h −1 ·mg of protein −1 respectively. The K m of the hydroxylase for m b 5 was 0.8 μM. Interestingly, m b 5 was not replaced with the soluble form of cytochrome b 5 , which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b 5 R (NADH-cytochrome b 5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and m b 5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of m b 5 by b 5 R. more...

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate. more...

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes. more...

ISP-1 (myriocin) is a potent inhibitor of serine palmitoyltransferase, the primary enzyme of sphingolipid biosynthesis, and is a useful tool for studying the biological functions of sphingolipids in both mammals and yeast (Saccharomyces cerevisiae). In a previous study, we cloned yeast multicopy suppressor genes for ISP-1, and one of these, YPK1/SLI2, was shown to encode a serine/threonine kinase which is a yeast homologue of mammalian SGK1 (serum/glucocorticoid-regulated kinase 1). In the present study, another gene, termed SLI1 (YGR212W; GenBank accession number CAA97239.1), was characterized. Sli1p has weak similarity to Atf1p and Atf2p, which are alcohol acetyltransferases. Although a sli1-null strain grew normally, the IC50 of ISP-1 for the growth of this strain was markedly decreased compared with that for the parental strain, indicating that Sli1p is a major contributor to ISP-1 resistance in yeast. On a sli1-null background, the increase in resistance to ISP-1 induced by YPK1 gene transfection was almost abolished. These data indicate that Sli1p co-operates with Ypk1p in mediating resistance to ISP-1 in yeast. Sli1p was found to convert ISP-1 into N-acetyl-ISP-1 in vitro. Furthermore, N-acetyl-ISP-1 did not share the ability of ISP-1 to inhibit the growth of yeast cells, and the serine palmitoyltransferase inhibitory activity of N-acetyl-ISP-1 was much lower than that of ISP-1. These data suggest that Sli1p inactivates ISP-1 due to its N-acetyltransferase activity towards ISP-1. more...

Cancer cells undergo distinct metabolic changes to cope with their hypoxic environment. These changes are achieved at least partly by the action of transcriptional factors called hypoxia-inducible factors (HIFs). We investigated gene expression in cultured human colon cancer cells induced by hypoxic conditions with special reference to cell-adhesion molecules and carbohydrate determinants having cell-adhesive activity by using DNA-microarray and RT-PCR techniques. Hypoxic culture of colon cancer cells induced a marked increase in expression of selectin ligands, the sialyl Lewis x and sialyl Lewis a determinants at the cell surface, which led to a definite increase in cancer cell adhesion to endothelial E-selectin. The transcription of genes for fucosyltransferase VII ( FUT7 ), sialyltransferase ST3Gal-I ( ST3O ), and UDP-galactose transporter-1 ( UGT1 ), which are all known to be involved in the synthesis of the carbohydrate ligands for E-selectin, was significantly induced in cancer cells by hypoxic culture. In addition, a remarkable induction was detected in the genes for syndecan-4 ( SDC4 ) and α5-integrin ( ITGA5 ), the cell-adhesion molecules involved in the enhanced adhesion of cancer cells to fibronectin. The transcriptional induction by hypoxia was reproduced in the luciferase-reporter assays for these genes, which were significantly suppressed by the co-transfection of a dominant-negative form of HIF. These results indicate that the metabolic shifts of cancer cells partly mediated by HIFs significantly enhance their adhesion to vascular endothelial cells, through both selectin- and integrin-mediated pathways, and suggest that this enhancement further facilitates hematogenous metastasis of cancers and tumor angiogenesis. more...

GM1 ganglioside-bound amyloid beta-protein (GM1-Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) plaques, has been suggested to accelerate amyloid fibril formation by acting as a seed. We have previously found using dye-labeled Abeta that Abeta recognizes a GM1 cluster, the formation of which is facilitated by cholesterol [Kakio, A., Nishimoto, S., Yanagisawa, K., Kozutsumi, Y., and Matsuzaki, K. (2001) J. Biol. Chem. 276, 24985-24990]. In this study, we investigated the ganglioside species-specificity in its potency to induce a conformational change of Abeta, by which ganglioside-bound Abeta acts as a seed for Abeta fibrillogenesis, using a major ganglioside occurring in brains (GM1, GD1a, GD1b, and GT1b) in raft-like membranes composed of cholesterol and sphingomyelin. Abeta recognized ganglioside clusters, the density of which increased with the number of sialic acid residues. Interestingly, however, mixing of gangliosides inhibited cluster formation. In contrast, the affinities of the protein for the clusters were similar irrespective of lipid composition and of the order of 10(6) M(-)(1) at 37 degrees C. Abeta underwent a conformational transition from an alpha-helix-rich structure to a beta-sheet-rich structure with the increase in protein density on the membrane. Ganglioside-bound Abeta proteins exhibited seeding abilities for amyloid formation. GM1-Abeta exhibited the strongest seeding potential, especially under beta-sheet-forming conditions. This study suggested that lipid composition including gangliosides and cholesterol strictly controls amyloid formation. more...

We have demonstrated that antibody-mediated crosslinking of GPI-anchored TAG-1 induced activation of src-family kinase Lyn and rapid tyrosine phosphorylation of an 80-kDa protein (p80), a putative substrate for Lyn, in the lipid raft fraction prepared from primary cerebellar cultures, suggesting the functional association of TAG-1 with Lyn in lipid rafts of the rat cerebellum. In this study, the association was confirmed using a cDNA expression system. TAG-1-expressing CHO transfectants exhibited enhanced self-aggregation and promoted neurite outgrowth of primary cerebellar cultures as a culture substrate. The anti-TAG-1 antibody co-immunoprecipitated Lyn with TAG-1 and induced co-patching of TAG-1 with Lyn in both TAG-1 and Lyn-expressing CHO transfectants. Density gradient analysis revealed that TAG-1 is present in the lipid raft fraction of the CHO transfectants. Furthermore, pretreatment with a sphingolipid biosynthesis inhibitor ISP-1 reduced the extent of tyrosine phosphorylation of p80 by the antibody-mediated crosslinking of TAG-1. Immunocytochemical study showed that both TAG-1 and Lyn are present in cerebellar granule cells. These observations suggest that TAG-1 associates with Lyn in lipid rafts of rat cerebellar granule cells. more...

In our previous study, the sphingosine-like immunosuppressant ISP-1 was shown to induce apoptosis in the mouse cytotoxic T cell line CTLL-2. In this study, we characterized the ISP-1-induced apoptotic pathway. Although caspase-3-like protease activity increases concomitantly with ISP-1-induced apoptosis in CTLL-2 cells, the apoptosis is not inhibited by caspase-3-like protease inhibitors, i.e. DEVD-cho and z-DEVD-fmk. In contrast, sphingosine-induced apoptosis in CTLL-2 cells is caspase-3-like protease-dependent. A caspase inhibitor with broad specificity, z-VAD-fmk, protects cells from apoptosis induced by ISP-1, indicating that ISP-1-induced apoptosis is dependent on caspase(s) other than caspase-3. Overexpression of Bcl-2 or Bcl-xL suppresses the apoptosis induced by ISP-1, although sphingosine-induced apoptosis is not efficiently inhibited by Bcl-2. Finally, ISP-1-induced mitochondrial depolarization, which is thought to be a checkpoint dividing the apoptotic pathway into upstream and downstream stages, is not inhibited by DEVD-cho, but is inhibited by z-VAD-fmk. These data suggest that a pathway dependent on caspase(s) other than caspase-3 is involved upstream of mitochondrial depolarization in ISP-1-induced apoptosis. more...

The mixed lineage kinase (MLK) family is a recently described protein kinase family. The MLKs contain a kinase domain followed by a dual leucine zipper-like motif. We previously reported the molecular cloning of LZK (leucine zipper-bearing kinase), a novel MLK, and that LZK activated the c-Jun NH2 terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway through MKK7 in cells. Here, we reveal that LZK forms dimers/oligomers through its dual leucine zipper-like motif, and that this is necessary for activation of the JNK/SAPK pathway. We also identify the C-terminal functional region of LZK, which is indispensable for the activation of SEK1, but not that of MKK7. more...

Recent reports have shown that sphingolipids are involved in several biological functions, including adhesion, differentiation, growth, and apoptosis (3, 22, 23, 25, 26, 44, 58). Ceramide and sphingosine-1-phosphate, which are metabolic products of sphingolipids, are thought to be upregulated by several stimuli and to serve as second messengers in signal transduction pathways (24, 27, 50, 55). Sphingosine-1-phosphate is upregulated by the platelet-derived growth factor stimulus (51), and the increase in sphingosine-1-phosphate promotes the growth of 3T3 fibroblast cells (66). Recently, G-protein-coupled cell surface receptors for sphingosine-1-phosphate were found in several cell lines (4, 40, 68). Furthermore, the possible involvement of intracellular sphingosine-1-phosphate in several signal transduction pathways has been postulated (31, 59). In addition to sphingosine-1-phosphate, intracellular ceramide upregulation through the sphingomyelin cycle on stimulation by tumor necrosis factor alpha (56), Fas ligand (11, 20, 21), or UV or γ irradiation (24) causes apoptosis of Jurkat T cells and several other cell lines, although exogenously added ceramide does not exert all of the downstream effects of these stimulants (30, 57, 64). The yeast Saccharomyces cerevisiae synthesizes sphingolipids similarly to mammalian sphingolipids except that phytosphingosine rather than sphingosine is the predominant sphingoid base (Fig. ​(Fig.1),1), and it lacks cell surface receptors for sphingosine-1-phosphate (16). Therefore, yeast is a useful system for studying the intracellular sphingolipid-mediated signaling pathway. Indeed, yeast has a sphingosine kinase which produces sphingosine-1-phosphate from sphingosine, and the former is a crucial substance involved in the respiration of yeast cells (39). Ceramide has been shown to induce G1 arrest in yeast via a ceramide-activated protein phosphatase (49). A new phosphatase, which modulates stress responses through sphingolipid metabolites, has also been isolated from yeast (42). However, most of the downstream pathway of intracellular sphingolipid-mediated signaling has not been well clarified in yeast or mammals. FIG. 1 Major sphingolipid biosynthetic pathways in yeast and mammalian cells. ISP-1 was isolated from Isaria sinclairii as a new type of potent immunosuppressant, the structure of which is homologous to that of sphingosine (17). In our previous studies, ISP-1 was found to inhibit mammalian serine palmitoyltransferase (SPT), which catalyzes the first step of sphingolipid biosynthesis. ISP-1 induced the apoptosis of mouse cytotoxic T cells (17, 45, 48) and rat Purkinje cells (18), which was triggered by reduction of the intercellular pools of sphingolipid metabolites. These results suggest that ISP-1 is a useful tool for the depletion of intracellular sphingolipids and for studying the sphingolipid-mediated signaling pathway in both mammals and yeast. We report that ISP-1 prevents yeast proliferation due to the inhibition of sphingolipid biosynthesis and that a multicopy suppressor gene of ISP-1 encodes a downstream kinase of the sphingolipid-mediated signaling pathway in yeast. more...

Our previous study revealed that compound 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcalpha2-6GalOMP) has the most promising affinity for mCD22. Replacing the subterminal galactose residue of 1 with benzyl or biphenylmethyl as aglycone led to 38- and 20-fold higher potency, respectively. This discovery represents a new direction in inhibitor design suitable for pharmaceutical development. more...

N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals, but it is not detectable in humans. The expression of NeuGc is controlled by cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase activity. We previously cloned a cDNA for mouse CMP-NeuAc hydroxylase and found that the human genome contains a homologue. We report here the molecular basis for the absence of NeuGc in humans. We cloned a cDNA for human CMP-NeuAc hydroxylase from a HeLa cell cDNA library. The cDNA encodes a 486-amino acid protein, and its deduced amino acid sequence lacks a domain corresponding to the N-terminal 104 amino acids of the mouse CMP-NeuAc hydroxylase protein, although the human protein is highly identical (93%) to the rest of the mouse hydroxylase protein. The N-terminal truncation of the human hydroxylase is caused by deletion of a 92-base pair-long exon in human genomic DNA. The human hydroxylase expressed in COS-7 cells exhibited no enzymatic activity, and a mouse hydroxylase mutant, which lacks the N-terminal domain, was also inactive. A chimera composed of the human hydroxylase and the N-terminal domain of the mouse hydroxylase displayed the enzyme activity. These results indicate that the human homologue of CMP-NeuAc hydroxylase is inactive because it lacks an N-terminal domain that is essential for enzyme activity. The absence of NeuGc in human glycoconjugates is due to a partial deletion in the gene that encodes CMP-NeuAc hydroxylase. more...

Fumonisin B1 (FB1) and aminopentol (AP1) (which is formed by hydrolysis of FB1) are found in corn contaminated with some strains of Fusarium moniliforme. Incubation of HT29 cells (a human colonic cell line) with FB1 or AP1 caused a significant reduction in cell number; AP1 was less potent, with 50 microM AP1 causing the same reduction (ca. 30% after 24 h) as 10 microM FB1. The reduction in cell number reflected increases in DNA fragmentation and the percentage of apoptotic cells. Both FB1 and AP1 caused the accumulation of sphinganine (25- and 35-fold by 10 microM FB1 and 50 microM AP1, respectively); thus, concentrations of FB1 and AP1 that caused comparable reductions in cell number were also similar with respect to elevation of sphinganine, a compound that is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with ISP-1 prevented the elevation in sphinganine, DNA fragmentation, and apoptosis induced by FB1. Therefore, these effects of FB1 on HT29 cells can be attributed to the accumulation of sphinganine. Since consumption of food contaminated with Fusarium moniliforme (Sheldon) exposes colonic cells to these mycotoxins, the possibility that FB1 and AP1 are toxic for intestinal cells in vivo should be evaluated, especially in the light of the recent report (Bhat et al., Clin. Toxicol. 35, 249, 1997) describing intestinal disturbances in humans after consumption of moldy corn and sorghum containing fumonisins. more...

Exogenous neutral glycosphingolipids induced homotypic cell-cell aggregation of a variety of hematopoietic cell lines. A mouse cytotoxic T cell line, CTLL-2, was chosen to study the neutral glycosphingolipid-induced aggregation. Among neutral glycosphingolipids tested, galactosylceramide (GalCer) and glucosylceramide (GlcCer) were potent inducers, followed by lactosylceramide (LacCer); globotriaosylceramide (Gb3Cer) and neolactotetraosylceramide (nLc4Cer) were less effective. GalCer that contained a non-hydroxy fatty acid was more efficient than GalCer containing an alpha-hydroxy fatty acid. The minimum concentration of GalCer containing a non-hydroxy fatty acid that induced aggregation was 1 microM, and maximum aggregation occurred at 10-20 microM within 24 h. Cytochalasin B and a mixture of sodium azide and 2-deoxyglucose inhibited the aggregation, whereas cycloheximide, actinomycin D, and colchicine did not. Aggregated and dispersed cells, which were designated as competent cells, re-aggregated in the absence of neutral glycosphingolipids. Anti-GalCer polyclonal antibody inhibited GalCer-induced reaggregation. Furthermore, competent cells bound and aggregated non-competent cells in the absence of neutral glycosphingolipids. Cell-cell aggregatign was similar for CTLL-2 cells and the other hematopoietic cells that were tested. These findings suggest that the neutral glycosphingolipid-induced cell-cell aggregation of CTLL-2 cells was mediated by heterophilic interaction(s) between glycosphingolipids and other cell surface components. These properties are shared by a variety of hematopoietic cell lines. more...

The HNK-1 carbohydrate epitope is characteristically expressed on a series of cell adhesion molecules and also on some glycolipids in the nervous system over a wide range of species from insect to mammal. The HNK-1 epitope is involved in cell–cell and/or cell–substrate interaction and recognition during the development of the nervous system. In this study, we isolated a novel glucuronyltransferase from rat brain, which is a key enzyme of the biosynthesis of the HNK-1 epitope on glycoproteins. Based on the partial amino acid sequences, we isolated cDNA encoding the glucuronyltransferase. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein with 347 amino acids and had no detectable similarity with any other proteins of known functions, including glucuronyltransferases of the liver and olfactory epithelium. Expression of a soluble recombinant form of the enzyme in COS-1 cells produced an active glucuronyltransferase. The selective expression of the glucuronyltransferase gene in the nervous system was consistent with the almost exclusive localization of the HNK-1 epitope in the nervous system. Transfection of the glucuronyltransferase cDNA into COS-1 cells induced not only expression of the HNK-1 epitope on the cell surface but also marked morphological changes of the cells, suggesting that the HNK-1 epitope associates with the cell–substratum interaction. more...

Delta12-Prostaglandin (PG) J2, a cyclopentenone prostaglandin, plays a role in various stress responses. BiP, a stress-inducible chaperone protein, is implicated in protein folding and translocation in endoplasmic reticulum and induced in the condition of accumulation of unfolded proteins. Here, we examined the effect of Delta12-PGJ2 on the expression of the BiP gene. Delta12-PGJ2 markedly stimulated the expression of the BiP gene in a time- and concentration-dependent manner in HeLa cells. This stimulation was specific for cyclopentenone PGs among various PGs. Cycloheximide pretreatment completely inhibited the Delta12-PGJ2-induced expression of the BiP gene, suggesting that the effects on nascent protein synthesis are involved in the signaling mechanism. Delta12-PGJ2 markedly stimulated the promoter activity of the 5'-flanking region of the BiP gene through the unfolded protein response element. Furthermore, Delta12-PGJ2 stimulated the enhancer activity of the 3'-half of the unfolded protein response element, and this stimulation required three nucleotides within this region. Gel mobility shift assay demonstrated that this region was occupied with two specific nuclear protein factors with different mobilities in the control cells, and Delta12-PGJ2 induced the dissociation of the protein-DNA complex with lower mobility. These findings indicate that Delta12-PGJ2 stimulates the expression of BiP gene through the 3'-half of the unfolded protein response element. more...

The serum-mannan binding protein (S-MBP) is a calcium-dependent C-type lectin specific for mannose and N-acetylglucosamine. S-MBP is known as a host defense factor involved in innate immunity, where the target ligands for S-MBP should be on the surface of exogenous microorganisms. In this study, we tried to find endogenous ligands for this endogenous lectin. Among the cells tested, only the lymphocytes from thymus of BALB/c mice expressed ligands for S-MBP on their surface, those from bone marrow, spleen, mesenteric lymph nodes and peripheral blood all being negative. Interestingly, among the thymocytes, only the immature thymocytes with the CD4+CD8+CD3low phenotype expressed ligands for S-MBP, and ligands for S-MBP decreased on their maturation. A major cell surface glycoprotein bearing S-MBP ligands was isolated and identified as CD45RO, which is a transmembrane protein with tyrosine phosphatase activity. Deglycosylation experiments with N-glycanase and endoglycosidase H indicated that the S-MBP ligands on thymic CD45 are high mannose type or hybrid type N-linked oligosaccharides. This unique presentation of S-MBP ligands on this special CD45 isoform suggested the possibility that the oligosaccharide portion of CD45 on immature thymocytes is associated with the maturation, development or selection events of thymocytes. more...

ISP-1/myriocin is a new type of remarkably potent immunosuppressant, the structure of which is homologous to sphingosine. ISP-1/myriocin inhibited the proliferation of an IL-2-dependent mouse cytotoxic T cell line, CTLL-2, at nanomole concentrations. ISP-1/myriocin inhibits serine palmitoyltransferase activity at picomole concentrations. This enzyme catalyzes the first step of sphingolipid biosynthesis and reduces the intracellular pool of sphingolipid intermediates. The growth inhibition induced by ISP-1/myriocin was completely abolished by the addition of sphingosines or sphingosine-1-phosphate, but not by sphingomyelin or glycosphingolipids. These results suggest that sphingosines or sphingosine-1-phosphate are associated with CTLL-2 proliferation, and ISP-1/myriocin suppresses T cell proliferation by the modulation of sphingolipid metabolism. ISP-1/myriocin should be a useful tool for the study of the sphingolipid pathway, which has been associated with various kinds of signal transduction. more...

We have proposed that CMP-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is mediated by an electron transport system consisting of cytochrome b5 (b5), b5 reducing factor(s), and CMP-NeuAc hydroxylase, all of which have been detected in the cytosolic fraction of mouse liver [Kozutsumi, Y., Kawano, T., Yamakawa, T., & Suzuki, A. (1990) J. Biochem. 108, 704-706]. In order to elucidate the reaction mechanism underlying CMP-NeuAc hydroxylation, the interaction between b5 and the hydroxylase was studied using a b5-immobilized affinity column. The enzyme activity was retarded on the b5 column in the presence of the substrate, CMP-NeuAc, but not in the presence of the reaction product, CMP-N-glycolyneuraminic acid (CMP-NeuGc). These findings suggest that the binding of CMP-NeuAc to CMP-NeuAc hydroxylase changes the conformation of the enzyme so as to construct a recognition site for b5, followed by the formation of a ternary complex through this domain. Then the transport of electrons from NAD(P)H to the enzyme through b5 takes place, CMP-NeuAc is converted to CMP-NeuGc, and finally the ternary complex dissociates into its components to release CMP-NeuGc. It is known that a soluble form of b5 is abundant in erythrocytes and is synthesized from a mRNA different from that for the microsomal form of b5. In order to determine the origin of b5 detected in the cytosolic fraction of mouse liver, the molecular forms of b5 mRNA expressed in mouse liver were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS) more...

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB). more...

Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected. more...

Human mannan-binding protein (MBP) is a C-type serum lectin involved in an immunoglobulin-independent host defense. Recently, a common defect of immune opsonin was found to be associated with very low serum levels of MBP. This deficiency was thought to be an effect of a single base substitution in the MBP gene, which resulted in the conversion of Gly54 to Asp. In this study, three mutant MBPs were expressed in COS-1 cells and the effects of these mutations were studied. Gly54Asp-MBP and Gly57Asp-MBP were secreted into the medium almost at the same levels as the wild type MBP, but the formation of higher multimers and the activation of complement were interfered with significantly. The other mutant, in which Leu was inserted into the Gly63-Gln-Gly sequence to restore the collagen motif of the Gly-Xaa-Yaa repeat, was secreted at levels similar to that of the wild type, formed higher multimers and activated complement in a manner not significantly altered from that of the wild type. These results are discussed with regard to the molecular basis of patients with opsonic defects. more...

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochrome b5. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochrome b5, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochrome b5, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression of N-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation. more...

In earlier studies, we identified the C-9 amido derivative 1 (9-(4′-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) and the C-9 amino derivative 2 (9-(4′-hydroxy-4-biphenyl)methylamino-9-deoxy-Neu5Gcα2-6GalOMP) have the most promising affinity for mouse CD22 and human CD22, respectively. Replacing the subterminal galactose residue (2-6Gal-OMP) of 1 with benzyl (5) or biphenylmethyl (6) as aglycone led to even higher potency for mCD22. In this study, both compounds showed improved potency and selectivity for CD22 (IC50 70 nM) and 712-fold more selective for CD22 than for MAG. The corresponding derivatives of 2, compounds 8 and 9, showed comparable activity to 2 but lower potency and selectivity than 5 and 6. Although compounds 5–9 are simple and small molecular weight antagonists, they showed much high potency and selectivity than the corresponding compounds having α 2-6Gal linkage. Both biological and computational docking simulation studies suggest that the 2-6Gal-OMP residues of 1 and 2 are not critical for binding process and could be replaced with hydrophobic non-carbohydrate moieties. The data presented herein has significant implications for the design and discovery of next-generation CD22-antagonists. more...

The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10–40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency. more...

Nitrogen starvation-mediated reduction of Ypk1 is suggested to suppress translational initiation, possibly in parallel with the target of rapamycin complex 1 (TORC1) signaling. However, the molecular mechanism that regulates Ypk1 in nitrogen-starved cells is poorly understood. Here we report that Ypk1 is a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system. Among various nutrient starvation methods used to elicit autophagy, rapid Ypk1 degradation was specific to nitrogen starvation. In screening genes required for such nitrogen starvation-specific vacuolar proteolysis, we found that autophagy-related degradation of Ypk1 depended on the endosomal sorting complex required for transport (ESCRT) machinery, which is conventionally thought to function in endosomal trafficking. In microscopic analyses, the disruption of ESCRT subunits resulted in the accumulation of both Ypk1 and autophagosomal Atg8 at a perivacuolar site that was distinct from conventional endosomes. ESCRT machinery was not involved in autophagic flux induced by the TORC1 inhibitor rapamycin, thus suggesting that ESCRT represents an exclusive mechanism of nitrogen starvation-specific proteolysis of Ypk1. Overall, we propose a novel regulation of Ypk1 that is specific to nitrogen limitation. more...

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G(i/o), whereas the SPC-induced calcium influx was dependent on G(q). Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G(i/o) and G(q). These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms. more...

The anti-CD20 monoclonal antibody (Ab) rituximab is accepted to be an effective therapeutic Ab for malignant B-cell lymphoma; however, discovery of other cell surface antigens is required for the option of antibody medicine. Considering that many tumor-associated antigens are glycans, we have searched glycoconjugates for the candidate antigens that therapeutic Abs target. To this end, we first focused on the difference in the glycogenes expression in terms of Epstein-Barr virus (EBV) infection of a Burkitt's lymphoma cell line, Akata. Using DNA array, flow cytometry and Western blotting, we found that Thy1 was highly expressed in EBV-positive Akata cells. Subsequently, Thy1 was found to be expressed in other B-cell lymphoma cell lines: BJAB, MutuI, and MutuIII, irrespective of EBV infection. Treatment of these cells with an anti-Thy1 monoclonal antibody inhibited proliferation more strongly than the therapeutic Ab rituximab. The B-cell lymphoma cell lines were classified based on the extent of the proliferation inhibition, which was not correlated with the expression level of Thy1. It is suggested that stable residence of receptor tyrosine kinases in lipid rafts sustains cell growth in B-cell lymphoma cells. more...

The generation of pigs devoid of Galα1,3Galβ1,4GlcNAc (Gal) residues has stimulated interest in non-Gal Ags as potentially important targets for Ab binding leading to rejection of pig organ xenografts in humans. Although N-glycolylneuraminic acid (NeuGc) epitopes, which are widely expressed on the endothelial cells of all mammals except humans, are likely targets of anti–non-Gal Abs, this aspect has not been investigated intensively owing to the absence of an appropriate animal model. In this study, we used CMAH−/− mice, which are completely deficient in NeuGc and thus produce anti-NeuGc Abs. Sera obtained from CMAH−/− mice and healthy human volunteers having anti-NeuGc Abs initiated complement-mediated lysis against CMAH+/+ cells in vitro. The cytotoxic activity of anti-NeuGc Abs was also determined in vivo (i.e., NeuGc-expressing CMAH+/+ mouse splenocytes that had been i.v. injected were completely eliminated in syngeneic CMAH−/− mice). CMAH−/− mice rejected the islets transplanted from syngeneic CMAH+/+ mice. Thus, the anti-NeuGc Ab-mediated response may be crucially involved in xenograft loss. This is the first direct demonstration of the immunogenic property of NeuGc determinants as targets of the corresponding Abs in CMAH+/+-to-CMAH−/− transplantation setting. more...

Siglec-2 is a mammalian sialic acid binding protein expressed on B-cell surfaces and is involved in the modulation of B-cell mediated immune response. We synthesized a unique starfish ganglioside, AG2 pentasaccharide Galfbeta(1-3)Galpalpha(1-4)Neu5Acalpha(2-3)Galpbeta(1-4)Glcp, and found that the synthetic pentasaccharide binds to human Siglec-2 by performing (1)H NMR experiments. Saturation transfer difference NMR experiments indicated that the C7-C9 side-chain and the acetamide moiety of the central sialic acid residue were located in the binding face of human Siglec-2. We determined the binding epitope of AG2 pentasaccharide to human Siglec-2, as the Galpalpha(1-4)Neu5Acalpha(2-3)Galp unit. more...

DNA microarray analysis enables us to provide a considerable amount of information on glyco-chain expression (Brazma et al. 2001; Ide et al. 2006). DNA microarray experiments are developed to obtain overall transcriptome of cells using DNAs spotted on a slide glass as probes and fluorescently labeled DNAs derived from mRNA samples. DNA microarray experiments require high-quality RNA. Thus, it is advisable to prepare RNA as intact as possible. The quality of RNA is usually assessed by the intactness of the peaks and the lack of degradation of RNA by capillary electrophoresis. This step is essential for the reliable final readout of microarray experiments. Following is the normal procedure operated in our laboratory for the DNA microarray experiments using RIKEN human and mouse glyco-chain-related DNA microarrays version 2 (human version: GEO platform GPL 3465 http://www.ncbi.nlm.nih.gov/geo/), which were designed by us and spotted by Takara Bio Inc. more...

The hydroxylation of CMP-N-acetylneuraminic acid (CMP-NeuAc) in the formation of CMP-N-glycolylneuraminic acid requires several components which comprise an electron transport system. A protein, which replaces one of the components, was purified to homogeneity from a horse erythrocyte lysate. Based on its partial amino acid sequence and immunological cross-reactivity, this protein was identified as soluble cytochrome b5 lacking the membrane domain of microsomal cytochrome b5. The electron transport system involved in CMP-NeuAc hydroxylation was reconstituted, and then characterized using the purified horse soluble cytochrome b5 and a fraction from mouse liver cytosol. The hydroxylation reaction requires a reducing reagent, DTT being the most effective. Either NADH or NADPH was used as an electron donor, but the activity with NADPH amounted to about 74% of that with NADH. The hydroxylation was inhibited by salts and azide due to interruption of the electron transport from NAD(P)H to cytochrome b5 and in the terminal enzyme reaction, respectively. more...

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1β (IL-1β), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1β expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression. more...

To understand physiological roles of tissue mast cells, we established a culture system where bone marrow-derived immature mast cells differentiate into the connective tissue-type mast cell (CTMC)-like cells through modifying the previous co-culture system with Swiss 3T3 fibroblasts. Our system was found to reproducibly mimic the differentiation of CTMCs on the basis of several criteria, such as granule maturation and sensitivity to cationic secretagogues. The gene expression profile obtained by the microarray analyses was found to reflect many aspects of the differentiation. Our system is thus helpful to gain deeper insights into terminal differentiation of CTMCs. more...

The galactosylsphingosine psychosine (Psy) is one of the sphingolipids and induce the formation of multinuclear cells in several cell lines by inhibiting cytokinesis. In the present report, we show that intracellular organelles, including wheat germ agglutinin (WGA)-positive vesicles and early endosomes, are selectively dispersed by Psy. WGA is a conventional Golgi marker and WGA-positive vesicles appeared to co-localize with the Golgi apparatus in untreated cells. Psy treatment induced the dispersal of WGA-positive vesicles without affecting the structure of the Golgi apparatus, resulting in discrimination of WGA-positive vesicles from the Golgi apparatus. In sharp contrast to this effect of Psy, WGA-positive vesicles were not affected by brefeldin A treatment, which induced the disappearance of the Golgi apparatus. Immunostaining with anti-TGN46 antibodies revealed that a large portion of the WGA-positive vesicles were derived from the trans-Golgi network. Notably, the dispersed WGA-positive vesicles did not stain with anti-syntaxin 6, another marker of the trans-Golgi network. During cytokinesis, WGA-positive vesicles in the cytoplasm decreased, and WGA staining accumulated at the cleavage furrow, which was apparently inhibited by the presence of Psy. These data suggest that the transport of WGA-positive vesicles to the cleavage furrow is associated with the progression of cytokinesis. more...