Ameliorating Effect of the Edible Mushroom Hericium erinaceus on Depressive-Like Behavior in Ovariectomized Rats.

Estrogen deficiency during menopause causes a variety of neurological symptoms, including depression. The edible Lion's Mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (HE), is a medicinal mushroom that has the potential for a neuroprotective effect and ameliorating neurological diseases, such as depression, anxiety, and neurodegenerative diseases. HE contains phytoestrogens, including daidzein and genistein. However, the ameliorating effect of HE on menopausal symptoms is not well understood. Here we investigated the impact of methanol extract of the HE fruiting body on depressive-like behavior in postmenopausal model rats. The activation of estrogen receptor alpha (ERα) causes body weight loss and uterine weight gain. Body weight gain and uterine weight loss by estrogen deficiency in ovariectomized (OVX) rats were reversed with 17ß-estradiol (E2) but not with HE. Thus, the phytoestrogens in HE may hardly activate ERα. Estrogen receptor beta (ERß) is expressed in the brain, and activation of ERß ameliorates menopausal depressive symptoms. Notably, depressive-like behavior in OVX rats evaluated in forced swim test was reduced by administration of not only E2 but also HE for 92 d. Long-term activation of ERα increases the risk of breast and uterine cancers. HE, therefore, may be effective in treating menopausal depression without the risk of carcinogenesis caused by ERα activation.

The Function of Sialidase Revealed by Sialidase Activity Imaging Probe.

Sialidase cleaves sialic acid residues from glycans such as glycoproteins and glycolipids. In the brain, desorption of the sialic acid by sialidase is essential for synaptic plasticity, learning and memory and synaptic transmission. BTP3-Neu5Ac has been developed for sensitive imaging of sialidase enzyme activity in mammalian tissues. Sialidase activity in the rat hippocampus detected with BTP3-Neu5Ac increases rapidly by neuronal depolarization. It is presumed that an increased sialidase activity in conjunction with neural excitation is involved in the formation of the neural circuit for memory. Since sialidase inhibits the exocytosis of the excitatory neurotransmitter glutamate, the increased sialidase activity by neural excitation might play a role in the negative feedback mechanism against the glutamate release. Mammalian tissues other than the brain have also been stained with BTP3-Neu5Ac. On the basis of information on the sialidase activity imaging in the pancreas, it was found that sialidase inhibitor can be used as an anti-diabetic drug that can avoid hypoglycemia, a serious side effect of insulin secretagogues. In this review, we discuss the role of sialidase in the brain as well as in the pancreas and skin, as revealed by using a sialidase activity imaging probe. We also present the detection of influenza virus with BTP3-Neu5Ac and modification of BTP3-Neu5Ac.

Mitigation of Memory Impairment in Ovariectomized Rats Using Garlic Powder Treated with Subcritical Water.

Women with estrogen deficiency are at the risk of suffering from neurological symptoms such as memory impairment. In the present study, we investigated the effect of garlic, Allium sativum L. (Asparagales: Amaryllidaceae), treated with subcritical water on memory impairment in ovariectomized (OVX) rats. OVX rats were administered garlic powder for 84 d. Hippocampus-dependent spatial memory was assessed using the Morris water maze test. Escape latency of the OVX rats increased compared with that of sham-operated rats. The prolonged escape latency of the OVX rats decreased to the level of that of sham-operated rats upon the administration of garlic powder (0.5% in feed). The weights of the body, uterus, and brain were not affected by the garlic powder administration. These results suggest that garlic powder treated with subcritical water mitigates memory impairment in OVX rats.

A I131V Substitution in the Fusion Glycoprotein of Human Parainfluenza Virus Type 1 Enhances Syncytium Formation and Virus Growth.

Human parainfluenza virus type 1 (hPIV1) has two spike glycoproteins, the hemagglutinin-neuraminidase (HN) glycoprotein as a receptor-binding protein and the fusion (F) glycoprotein as a membrane-fusion protein. The F glycoprotein mediates both membrane fusion between the virus and cell and membrane fusion between cells, called syncytium formation. Wild-type C35 strain (WT) of hPIV1 shows little syncytium formation of infected cells during virus growth. In the present study, we isolated a variant virus (Vr) from the WT that showed enhanced syncytium formation of infected cells by using our previously established hPIV1 plaque formation assay. Vr formed a larger focus and showed increased virus growth compared with WT. Sequence analysis of the spike glycoprotein genes showed that the Vr had a single amino acid substitution of Ile to Val at position 131 in the fusion peptide region of the F glycoprotein without any substitutions of the HN glycoprotein. The Vr F glycoprotein showed enhanced syncytium formation in F and HN glycoprotein-expressing cells. Additionally, expression of the Vr F glycoprotein increased the focus area of the WT-infected cells. The single amino acid substitution at position 131 in the F glycoprotein of hPIV1 gives hPIV1 abilities to enhance syncytium formation and increase cell-to-cell spread. The present study supports the possibility that hPIV1 acquires increased virus growth in vitro from promotion of direct cell-to-cell transmission by syncytium formation.

Rapid regulation of sialidase activity in response to neural activity and sialic acid removal during memory processing in rat hippocampus.

Sialidase cleaves sialic acids on the extracellular cell surface as well as inside the cell and is necessary for normal long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses and for hippocampus-dependent spatial memory. Here, we investigated in detail the role of sialidase in memory processing. Sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac) or 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB was increased by high-K+-induced membrane depolarization. Sialidase activity was also increased by chemical LTP induction with forskolin and activation of BDNF signaling, non-NMDA receptors, or NMDA receptors. The increase in sialidase activity with neural excitation appears to be caused not by secreted sialidase or by an increase in sialidase expression but by a change in the subcellular localization of sialidase. Astrocytes as well as neurons are also involved in the neural activity-dependent increase in sialidase activity. Sialidase activity visualized with a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe for sialidase activity, at the CA3 stratum lucidum of rat acute hippocampal slices was immediately increased in response to LTP-inducible high-frequency stimulation on a time scale of seconds. To obtain direct evidence for sialic acid removal on the extracellular cell surface during neural excitation, the extracellular free sialic acid level in the hippocampus was monitored using in vivo microdialysis. The free sialic acid level was increased by high-K+-induced membrane depolarization. Desialylation also occurred during hippocampus-dependent memory formation in a contextual fear-conditioning paradigm. Our results show that neural activity-dependent desialylation by sialidase may be involved in hippocampal memory processing.

Substrate Specificity of Equine and Human Influenza A Virus Sialidase to Molecular Species of Sialic Acid.

Most equine influenza A viruses (IAVs) show strong binding to glycoconjugates containing N-glycolylneuraminic acid (Neu5Gc) as well as N-acetylneuraminic acid (Neu5Ac). Therefore, the progeny of equine IAV is thought to be released from the infected cell surface through removal of sialic acids by the viral sialidase. In the present study, equine IAV sialidases showed significantly lower substrate affinity than that of human IAV sialidases to artificial and natural Neu5Gc-conjugated substrates. The substrate specificity of equine IAV sialidases is in disagreement with their binding specificity to molecular species of sialic acid. The results suggest that substrate specificity of equine IAV sialidase for Neu5Ac, rather than for Neu5Gc, is important for an advantage at the early infection stage and the process of progeny virus release from the surface of infected cells.

Functional analysis of glyco-molecules that bind with influenza virus.

Influenza A virus (IAV) recognizes terminal sialic acid of sialoglyco-conjugates on host cells through the viral envelope glycoprotein hemagglutinin (HA), followed by initiation of entry into the cells. Molecular species of sialic acid are largely divided into two moieties: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). A receptor for IAV infection generally means Neu5Ac. Almost all equine IAVs and some human, swine, and duck IAVs bind not only to Neu5Ac but also to Neu5Gc. In nonhuman animals, Neu5Gc has been detected in swine and equine tracheas and the duck colon, which are the main replication sites of mammalian and avian IAVs. Therefore, Neu5Gc in these sites has been suggested to be a functional receptor for IAV infection. Humans cannot synthesize Neu5Gc due to a genetic defect of the Neu5Gc-synthesizing enzyme. We evaluated the receptor function of Neu5Gc in IAV infection in human cells. Our results indicated that Neu5Gc expression on the surface of human cells is not a functional receptor for IAV infection and that it has a negative effect on infectivity of IAV possessing Neu5Gc binding ability. IAV also binds to non-sialo 3-O-sulfated galactosylceramide (sulfatide). Sulfatide has been suggested to be a functional receptor for IAV infection. However, we have shown that sulfatide is not a functional receptor for IAV infection and that the binding of HA with sulfatide enhances progeny virus production. It is expected that functions of these glyco-molecules can be used in prevention and development of new drugs against IAV.

N-glycolylneuraminic acid on human epithelial cells prevents entry of influenza A viruses that possess N-glycolylneuraminic acid binding ability.

UNLABELLED: Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. IMPORTANCE: Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.

Consecutive oral administration of Bifidobacterium longum MM-2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model.

Bifidobacterium, one of the major components of intestinal microflora, shows anti-influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM-2 (MM-2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM-2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro-inflammatory cytokines such as IL-6 and TNF-α in bronchoalveolar lavage fluid. The anti-IFV mechanism of MM-2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN-γ, IL-2, IL-12 and IL-18. Even in non-infected mice, MM-2 administration also induced significant enhancement of both IFN-γ production by Peyer's patch cells (PPs) and splenetic NK cell activity. Oral administration of MM-2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti-IFV effect. MM-2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.

Role of sulfatide in influenza A virus replication.

Sulfatide is a 3-O-sulfated galactosylceramide that is abundantly expressed in the gastrointestinal tract, kidney, trachea, and particularly the central nervous system. Cellular sulfatide is mainly localized in the Golgi apparatus, cellular membrane, and lysosomes in cytosol. Since our earlier report showed that the influenza A virus specifically binds to sulfatide, we have investigated the roles of sulfatide in the influenza A virus lifecycle. The viral binding is independent of sialic acids, which function as virus receptors in virus attachment to the host cell surface. Sulfatide is recognized by the ectodomain of the viral envelope glycoprotein hemagglutinin (HA). Nascent HA is transported on the surface membrane of infected cells. The binding of HA with sulfatide on the cell surface induces apoptosis through potential loss of the mitochondrial membrane and nuclear translocation of apoptosis-inducing factor in mitochondria, where PB1-F2 peptide from the viral gene is accumulated. In the nucleus of infected cells, viral ribonucleoprotein (vRNP) complexes are formed from viral RNA genomes, viral nucleoprotein, and viral RNA polymerase subunits, and these complexes are selectively exported into cytosol through the nuclear membrane. The apoptosis significantly enhances the nuclear export of vRNP complexes, resulting in efficient formation of progeny viruses and facilitation of virus replication. At that time, activation of the Raf/mitogen-activated protein extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway through sulfatide is associated with virus replication. Our studies have demonstrated that sulfatide is not a viral receptor for virus infection, and that the binding of HA with sulfatide functions as an initiation switch for the formation of progeny viruses.

Low-pH Stability of Influenza A Virus Sialidase Contributing to Virus Replication and Pandemic.

The spike glycoprotein neuraminidase (NA) of influenza A virus (IAV) has sialidase activity that cleaves the terminal sialic acids (viral receptors) from oligosaccharide chains of glycoconjugates. A new antigenicity of viral surface glycoproteins for humans has pandemic potential. We found "low-pH stability of sialidase activity" in NA. The low-pH stability can maintain sialidase activity under acidic conditions of pH 4-5. For human IAVs, NAs of all pandemic viruses were low-pH-stable, whereas those of almost all human seasonal viruses were not. The low-pH stability was dependent on amino acid residues near the active site, the calcium ion-binding site, and the subunit interfaces of the NA homotetramer, suggesting effects of the active site and the homotetramer on structural stability. IAVs with the low-pH-stable NA showed much higher virus replication rates than those of IAVs with low-pH-unstable NA, which was correlated with maintenance of sialidase activity under an endocytic pathway of the viral cell entry mechanism, indicating contribution of low-pH stability to high replication rates of pandemic viruses. The low-pH-stable NA of the 1968 H3N2 pandemic virus was derived from the low-pH-stable NA of H2N2 human seasonal virus, one of two types classified by both low-pH stability in N2 NA and a phylogenetic tree of N2 NA genes. The 2009 H1N1 pandemic virus acquired low-pH-stable NA by two amino acid substitutions at the early stage of the 2009 pandemic. It is thought that low-pH stability contributes to infection spread in a pandemic through enhancement of virus replication.

Production and Purification of Secretory Simian Cytidine Monophosphate-N-acetylneuraminic Acid Hydroxylase Using Baculovirus-Protein Expression System.

Cytidine monophosphate (CMP) N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) is an essential enzyme for N-glycolylneuraminic acid (Neu5Gc) synthesis. In humans, Neu5Gc cannot be synthesized because of a deletion in the CMAH gene. Since Neu5Gc research has not been actively performed in comparison with Neu5Ac research, little is known about the function of Neu5Gc. Possible reasons are that CMAH for controlling Neu5Gc synthesis is not understood well at the molecular level, that commercial Neu5Gc is expensive, and that addition of exogenous Neu5Gc to glycoconjugates is not a general method because of the difficulty in obtaining CMAH. One solution to these problems is to achieve large-scale production of CMAH with enzymatic activity. To produce and purify CMAH as simply as possible, we generated simian CMAH as a secretory protein with a histidine tag using a baculovirus protein expression system. After culture of baculovirus-infected cells in serum-free medium, secretory simian CMAH (approximately 180 µg) was highly purified from the supernatant (150 mL) of cell culture. HPLC analysis showed conversion of CMP-Neu5Ac to CMP-Neu5Gc by the secretory CMAH. We succeeded in producing secretory CMAH with enzymatic activity that is easy to purify. In addition, peptide-N-glycosidase F treatment of CMAH indicated that secretory CMAH was a glycoprotein with N-glycan. It will also contribute to research on Neu5Gc function by easy-to-use methods for controlling Neu5Gc synthesis, for exogenous addition of Neu5Gc to glycoconjugates and by application to industrial Neu5Gc synthesis.

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses.

Human parainfluenza virus type 1 (hPIV1) does not form clear plaque by the conventional plaque formation assay because of slightly a cytopathic effects in many cell lines infected with hPIV1, thus making in virus titration, isolation and inhibitor evaluation difficult. We have succeeded in fluorescent histochemical visualization of sialidase activities of influenza A and B viruses, Newcastle disease virus and Sendai virus by using a novel fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). In this study, we applied the BTP3-Neu5Ac assay for rapid detection of hPIV1 and hPIV type 3. The BTP3-Neu5Ac assay could histochemically visualize dot-blotted hPIVs on a membrane and hPIV-infected cells as local fluorescence under UV irradiation. We succeeded in distinct fluorescent visualization of hPIV1-infected cells in only 3 d using the BTP3-Neu5Ac assay. Due to there being no fixation, hPIV1 was isolated directly from fluorescent stained focus cells by the BTP3-Neu5Ac assay. Establishment of a sensitive, easy, and rapid fluorescent focus detection assay for hPIV, hPIV1 in particular will contribute greatly to progress in hPIV studies.

Histochemical imaging of alkaline phosphatase using a novel fluorescent substrate.

Histochemical visualization of phosphatase is exclusively required for Western immunoblotting and antigen-positive cell staining using an alkaline phosphatase (AP)-labeled secondary antibody. This detection has been performed by several reagents including 5-bromo-4-chloro-3-indolyl-phosphate (X-Phos), nitro blue tetrazolium (NBT), 3-(2'-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane and 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-[3H]-quinazolinone (ELF® 97 Phosphate). We previously reported that 2-(benzothiazol-2-yl)-4-bromophenol bonded with N-acetylneuraminic acid (BTP3-Neu5Ac), enabled fluorescent histochemical visualization of sialidase activity. 2-(Benzothiazol-2-yl)-4-bromophenol (BTP3), which is formed from BTP3-Neu5Ac by sialidase reaction, is a crystalline, insoluble and stable fluorogenic compound, deposited at the site of enzyme activity. We developed a BTP3 phosphate ester (BTP3-Phos) for the purpose of fluorescent histochemical visualization of phosphatase activity. BTP3-Phos emitted fluorescence in a manner dependent on the concentration of the AP-labeled antibody. BTP3-Phos also enabled fluorescent histochemical visualization of AP-blotted dots in a manner dependent on the concentration of the AP-labeled antibody. The detection sensitivity of BTP3-Phos was estimated to be greater than that of the conventional method using X-Phos and NBT. Influenza A virus-infected cells were fixed and reacted with anti-influenza A virus antibodies and incubated continuously with an AP-labeled secondary antibody. BTP3-Phos stained the infected cells with distinct green fluorescence. These results indicate that BTP3-Phos can enable fluorescent immunohistochemical staining analysis using an AP-labeled antibody. BTP3-Phos would be beneficial for histochemical staining of AP activity, and may be applicable for multi-color staining or a cell sorter.

Visualizing intracellular sialidase activity of influenza A virus neuraminidase using a fluorescence imaging probe.

In influenza A virus-infected cells, newly synthesized viral neuraminidases (NAs) transiently localize at the host cell Golgi due to glycosylation, before their expression on the cell surface. It remains unproven whether Golgi-localized intracellular NAs exhibit sialidase activity. We have developed a sialidase imaging probe, [2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenyl]-α-D-N-acetylneuraminic acid (BTP9-Neu5Ac). This probe is designed to be cleaved by sialidase activity, resulting in the release of a hydrophobic fluorescent compound, 2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenol (BTP9). BTP9-Neu5Ac makes the location of sialidase activity visually detectable by the BTP9 fluorescence that results from the action of sialidase activity. In this study, we established a protocol to visualize the sialidase activity of intracellular NA at the Golgi of influenza A virus-infected cells using BTP9-Neu5Ac. Furthermore, we employed this fluorescence imaging protocol to elucidate the intracellular inhibition of laninamivir octanoate, an anti-influenza drug. At approximately 7 h after infection, newly synthesized viral NAs localized at the Golgi. Using our developed protocol, we successfully histochemically stained the sialidase activity of intracellular viral NAs localized at the Golgi. Importantly, we observed that laninamivir octanoate effectively inhibited the intracellular viral NA, in contrast to drugs like zanamivir or laninamivir. Our study establishes a visualization protocol for intracellular viral NA sialidase activity and visualizes the inhibitory effect of laninamivir octanoate on Golgi-localized intracellular viral NA in infected cells.

VP1 of human and murine noroviruses recognizes glycolipid sulfatide via the P domain.

Noroviruses are a prevalent cause of human viral gastroenteritis, yet the precise mechanisms underlying their infection cycle, particularly their interactions with and entry into cells, remain poorly understood. Human norovirus (HuNoV) primarily targets human small intestinal epithelial cells, within which 3-O sulfogalactosylceramide (sulfatide) ranks among the most abundant glycosphingolipids (GSLs). While sulfatide involvement in the binding and infection mechanism of several viruses has been documented, its interaction with noroviruses remains underexplored. This study investigated whether noroviruses interact with sulfatide. We found that the recombinant viral capsid protein VP1 of HuNoV (genogroups I and II) and murine norovirus (genogroup V) exhibited robust binding to sulfatide compared with other tested GSLs using ELISA, TLC binding assay, and qRT-PCR binding assay. Notably, we found that sulfatide is a novel binding target for norovirus particles. Overall, our findings reveal a previously unknown norovirus-sulfatide interaction, proposing sulfatide as a potential candidate for norovirus infection receptors.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

Binding kinetics of sulfatide with influenza A virus hemagglutinin.

Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k on) of 1.41 × 10(4) M(-1) sec(-1), dissociation rate constant (k off) of 2.03 × 10(-4) sec(-1) and K d of 1.44 × 10(-8) M to sulfatide immobilized on a sensor chip. The k off values of sHA were similar for sulfatide and GD1a, whereas the k on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.

2-(Benzothiazol-2-yl)-phenyl-ß-d-galactopyranoside derivatives as fluorescent pigment dyeing substrates and their application for the assay of ß-d-galactosidase activities.

2-(Benzothiazol-2-yl)-phenyl-ß-d-galactopyranoside derivatives were synthesized as novel artificial fluorescent pigment dyeing substrates for ß-d-galactosidase. The substrates, which exhibited non-fluorescence or weak fluorescence in solution phase, were smoothly hydrolyzed by ß-d-galactosidase from Aspergillus oryzae and yielded a water-insoluble strong fluorescent pigment. The difference of fluorescent intensity exhibited a linear relationship with the amount of enzyme.

Syntheses of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified by α-acylaminoamido groups at the C-4 position using isocyanide-based four-component coupling and biological evaluation as inhibitors of human parainfluenza virus type 1.

Novel sialidase inhibitors 11 having an α-acylaminoamido group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using one-pot isocyanide-based four-component condensation, and their inhibitory activities against hPIV-1 sialidase were studied. Compound 11b showed inhibitory activity (IC(50)=5.1 mM) against hPIV-1 sialidase. The degree of inhibition of 11b was much weaker than that of 1 (IC(50)=0.3 mM).