Oseltamivir (Tamiflu) and zanamivir (Relenza), two extensively used clinically effective anti-influenza drugs, are viral sialidase (also known as neuraminidase) inhibitors that prevent the release of progeny virions and thereby limit the spread of infection. Recently mortalities and neuropsychiatric events have been reported with the use of oseltamivir, especially in pediatric cases in Japan, suggesting that these drugs might also inhibit endogenous enzymes involved in sialic acid metabolism, including sialidase, sialyltransferase, and CMPsynthase, in addition to their inhibitory effects on the viral sialidase. The possible inhibition could account for some of the rare side effects of oseltamivir. However, there has been little direct evidence in regard to the sensitivities of animal sialidases to these drugs. Here, we examined whether these inhibitors might indeed affect the activities of human sialidases, which differ in primary structures and enzyme properties but possess tertiary structures similar to those of the viral enzymes. Using recombinant enzymes corresponding to the four human sialidases identified so far, we found that oseltamivir carboxylate scarcely affected the activities of any of the sialidases, even at 1 mM, while zanamivir significantly inhibited the human sialidases NEU3 and NEU2 in the micromolar range (K i , 3.7 ؎ 0.48 and 12.9 ؎ 0.07 M, respectively), providing a contrast to the low nanomolar concentrations at which these drugs block the activity of the viral sialidases.

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19 -38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both ␣233 and ␣238 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.

Based on the human cDNA sequence predicted to represent the NEU4 sialidase gene in public databases, a cDNA covering the entire coding sequence was isolated from human brain and expressed in mammalian cells. The cDNA encodes two isoforms: one possessing an N-terminal 12-amino-acid sequence that is predicted to be a mitochondrial targeting sequence, and the other lacking these amino acids. Expression of the isoforms is tissue specific, as assessed by reverse transcription-PCR. Brain, muscle and kidney contained both isoforms; liver showed the highest expression, and the short form was predominant in this organ. In transiently transfected COS-1 cells, enzyme activity was markedly increased with gangliosides as well as with glycoproteins and oligosaccharides as substrates compared with the control levels. This differs from findings with other human sialidases. Although the isoforms were not distinguishable with regard to substrate specificity, they exhibited differential subcellular localizations. Immunofluorescence microscopy and biochemical fractionation demonstrated that an exogenously expressed haemagglutinin-tagged long form of NEU4 was concentrated in mitochondria in several human culture cell types, whereas the short form was present in intracellular membranes, indicating that the sequence comprising the N-terminal 12 amino acid residues acts as a targeting signal for mitochondria. Co-localization of the long form to mitochondria was further supported by efficient targeting of the N-terminal region fused to enhanced green fluorescent protein, and by the targeting failure of a mutant with an amino acid substitution in this region. NEU4 is possibly involved in regulation of apoptosis by modulation of ganglioside G(D3), which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.

Novel technologies to establish three-dimensional, in vivo-like tissue consisting of various types of cells are required for tissue engineering. We applied magnetic force to construct a heterotypic, layered coculture system of rat hepatocytes and human aortic endothelial cells (HAECs) that was not limited by cell type. Magnetite cationic liposomes carrying a positive surface charge to improve adsorption accumulated in HAECs at a concentration of 38 pg of magnetite per cell. Magnetically labeled HAECs specifically accumulated onto hepatocyte monolayers at sites where a magnet (4000 G) was positioned, and then adhered to form a heterotypic, layered construct with tight and close contact. This cocultured construct significantly (p < 0.05) enhanced albumin secretion by hepatocytes compared with that in homotypic cultures of hepatocytes or heterotypic cocultures of hepatocytes and HAECs without magnets. These results suggest that this novel use of magnetite nanoparticles and magnetic force, which we refer to as "magnetic force-based tissue engineering" (Mag-TE), offers a major advancement in tissue engineering.

Novel technologies to establish three-dimensional constructs are desired for tissue engineering. In the present study, magnetic force was used to construct multilayered keratinocyte sheets and harvest the sheets without enzymatic treatment. Our original magnetite cationic liposomes, which have a positive surface charge in order to improve adsorption, were taken up by human keratinocytes at a concentration of 33 pg of magnetite per cell. The magnetically labeled keratinocytes (2x10(6) cells, which corresponds to 5 times the confluent concentration against the culture area of 24-well plates, in order to produce 5-layered keratinocyte sheets) were seeded into a 24-well ultralow-attachment plate, the surface of which was composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. A magnet (4000 G) was placed under the well, and the keratinocytes formed a five-layered construct in low-calcium medium (calcium concentration, 0.15 mM) after 24 h of culture. Subsequently, when the five-layered keratinocytes were cultured in high-calcium medium (calcium concentration, 1.0 mM), keratinocytes further stratified, resulting in the formation of 10-layered epidermal sheets. When the magnet was removed, the sheets were detached from the bottom of the plates, and the sheets could be harvested with a magnet. These results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach for tissue engineering.

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Genomic imprinting in mammals marks the two parental alleles resulting in differential gene expression. Imprinted loci are characterized by distinct epigenetic modifications such as differential DNA methylation and asynchronous replication timing. To determine the role of DNA methylation in replication timing of imprinted loci, we analyzed replication timing in Dnmt1-and Dnmt3L-deficient embryonic stem (ES) cells, which lack differential DNA methylation and imprinted gene expression. Asynchronous replication is maintained in these ES cells, indicating that asynchronous replication is parent-specific without the requirement for differential DNA methylation. Imprinting centers are required for regional control of imprinted gene expression. Analysis of replication fork movement and three-dimensional RNA and DNA fluoroscent in situ hybridization (FISH) analysis of the Igf2-H19 locus in various cell types indicate that the Igf2-H19 imprinting center differentially regulates replication timing of nearby replicons and subnuclear localization. Based on these observations, we suggest a model in which cis elements containing nonmethylation imprints are responsible for the movement of parental imprinted loci to distinct nuclear compartments with different replication characteristics resulting in asynchronous replication timing.[Keywords: Asynchronous replication; genomic imprinting; nuclear localization; DNA methylation; Igf2-H19; replication fork] Genomic imprinting is an epigenetic mechanism that marks the two parental alleles during gametogenesis, resulting in differential gene expression in the developing embryo and adult. The epigenetic modifications responsible for establishing and maintaining imprinted gene expression are thought to involve DNA methylation, chromatin modifications, and asynchronous replication timing. For several imprinted loci specific cis control elements, imprinting centers (ICs), have been identified which are required for regional control of imprinting, imprinted gene expression, and asynchronous replication timing. All identified ICs harbor regions that contain differentially methylated DNA sequences. These methylation imprints are established in the germline and are resistant to global demethylation during the early cleavages of the preimplantation embryo. To date, DNA methylation is the only epigenetic mark shown to be required for the maintenance of imprinted gene expression. Embryos deficient for the DNA maintenance methyl transferase gene, Dnmt1, lack imprints and die early during development (Li et al. 1992). Moreover, embryos derived from females deficient for Dnmt3L, a newly identified de novo DNA methyl transferase gene that lacks the catalytic domain shared by the other DNA methyl transferases, were shown to lack maternal imprints and fail to develop past 9.5 days postcoitum (dpc; Bourc'his et al. 2001;Hata et al. 2002). A similar phenotype was observed in embryos generated by fertilization of oocytes deficient for the de novo DNA methyl transferase Dnmt3a, suggesting that these prot...

Aberrant sialylation in cancer cells is thought to be a characteristic feature associated with malignant properties including invasiveness and metastatic potential. Sialidase which catalyzes the removal of sialic acid residues from glycoproteins and glycolipids, has been suggested to play important roles in many biological processes through regulation of cellular sialic acid contents. The altered expression of sialidase observed in cancer would, therefore, suggest its involvement in the malignant process. In mammalian cells, three types of sialidase cloned and characterized to date were found to behave in different manners during carcinogenesis. Recent progress in molecular cloning of these sialidases has facilitated elucidation of the molecular mechanisms and significance of these alterations. Herein we briefly describe our own studies on sialidase changes associated with malignant transformation and summarize the topic from both a retrospective and a prospective viewpoint. Sialidases are indeed closely related to malignancy and are thus potential targets for cancer diagnosis and therapy.