Asymmetric dimethylarginine accumulation under hyperglycemia facilitates ß-cell apoptosis via inhibiting nitric oxide production.

Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) has been shown to suppress apoptosis in pancreatic ß-cells. In the present study, the influence of asymmetric dimethylarginine (ADMA), the major endogenous inhibitor of NOS, on the apoptosis-suppressive effect of NO was investigated. The expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2), an ADMA-metabolizing enzyme, in INS-1 ß-cells and in mouse pancreatic islets was drastically reduced by in vitro exposure to high-concentration glucose (20 mM) and by in vivo treatment of mice with the insulin receptor blocker S661, which resulted in hyperglycemia, respectively. In line with this, a higher ADMA level was observed in INS-1 cells exposed to 20 mM glucose. The treatment of INS-1 cells with ADMA, similarly to with the NOS inhibitor NG-nitro-L-arginine methyl ester, significantly facilitated 20 mM glucose-induced increase in cleaved caspase-3 protein expression. Furthermore, increased protein expression of cleaved caspase-3 and CHOP was observed in INS-1 cells with knockdown of DDAH2. These results suggest that ADMA accumulation through a decrease in DDAH2 expression in ß-cells, which is induced under hyperglycemic conditions, facilitates ß-cell apoptosis through suppression of cNOS-mediated NO production.

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.

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

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.

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.

Young Coconut Juice Supplementation Results in Greater Bone Mass and Bone Formation Indices in Ovariectomized Rats: A Preliminary Study.

Young coconut juice (Cocos nucifera Linn.) (YCJ) has traditionally been consumed to alleviate symptoms associated with menopause by women in Southeast Asia. The aim of the present study was to determine the effects of YCJ on bone metabolism in ovariectomized rats. Female 10-week-old Wistar rats were randomly assigned to the following 4 groups: Baseline, Sham, Ovx, and Ovx + YCJ (n = 10 rats per group). Rats in the Baseline group were sacrificed immediately, and those in the other groups were subjected to either sham operation (Sham) or bilateral ovariectomy (Ovx and Ovx + YCJ). The Ovx + YCJ rats were administered 5×-concentrated YCJ at a dose of 10 mL/kg body weight per day. Six weeks after surgery, the rats were sacrificed, and indices of bone mass and bone histomorphometry were measured. The bone mineral density of the left femur was significantly higher in the Ovx + YCJ group compared with the Ovx group. In addition, the Ovx + YCJ group showed significantly higher measurements for bone formation rate compared with the Ovx group. These findings suggest that YCJ supplementation has a positive effect on bone metabolism and thus represents a possible intervention to slow the bone loss observed following menopause.

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.

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.

Improvement of depression-like behavior and memory impairment with the ethanol extract of Pleurotus eryngii in ovariectomized rats.

Ethanol extract of Pleurotus eryngii (DC.) QUÉL has estrogen-like activities that protect against bone loss caused by estrogen deficiency. In the present study, we investigated the effect of P. eryngii on depression-like behavior and memory impairment in ovariectomized (OVX) rats. Immobility time during a forced swimming test was significantly longer for OVX rats than for sham-operated rats. The depression-like behavior in OVX rats was improved by long-term administration of the ethanol extract of P. eryngii (500 mg/kg body weight (b.w.)/d). Spatial memory impairment in OVX rats assessed by the Morris water maze test was also improved by P. eryngii extract without any effect on motility. These results suggested that P. eryngii extract has estrogen-like improvement activity against depression-like behavior and memory impairment in OVX rats. Additionally, increase in the amount of synaptosomal zinc after ovariectomy was inhibited by P. eryngii extract. Since zinc in synaptic vesicles is important for memory function and is linked to the pathophysiology of depression, normalization of zinc signaling would be involved in the beneficial effect of P. eryngii extract on neurological disorders after ovariectomy.

Effects of 10-Hydroxy-2-decenoic Acid and 10-Hydroxydecanoic Acid in Royal Jelly on Bone Metabolism in Ovariectomized Rats: A Pilot Study.

Although previous studies have demonstrated that royal jelly (RJ) may have estrogenic properties and prevent postmenopausal bone loss, the underlying mechanisms are not fully understood. This animal study aimed to investigate the effects of specific fatty acids of RJ, 10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), in ovariectomized rats. Ten-week-old female Wistar rats were divided into the Baseline, Sham, Ovx, Ovx + 10H2DA, and Ovx + 10HDAA groups. Rats in the Baseline group were sacrificed immediately, whereas those in the other groups were subjected to either a sham operation or bilateral ovariectomy. The animals in the Ovx + 10H2DA and Ovx + 10HDAA groups were fed diets containing 10H2DA and 10HDAA, respectively. Twelve weeks after surgery, the rats were sacrificed, and indices of bone mass and bone mechanics were analyzed. Femoral bone mineral density was significantly lower in the Ovx group than in the Sham group (p < 0.01). Administration of 10H2DA or 10HDAA did not ameliorate bone loss after ovariectomy. In addition, administration of these fatty acids diminished femur bone stiffness in ovariectomized rats (p < 0.01 and p < 0.05, respectively). These findings suggest that the favorable effects of RJ may not be exerted solely by 10H2DA or 10HDAA. However, these effects may be exhibited in combination with other RJ constituents.

Allyl Isothiocyanate Maintains DHA-Containing Glycerophospholipids and Ameliorates the Cognitive Function Decline in OVX Mice.

Low-temperature-induced fatty acid desaturation is highly conserved in animals, plants, and bacteria. Allyl isothiocyanate (AITC) is an agonist of the transient receptor potential ankyrin 1 (TRPA1), which is activated by various chemophysiological stimuli, including low temperature. However, whether AITC induces fatty acid desaturation remains unknown. We showed here that AITC increased levels of glycerophospholipids (GP) esterified with unsaturated fatty acids, especially docosahexaenoic acid (DHA) in TRPA1-expressing HEK cells. Additionally, GP-DHA including phosphatidylcholine (18:0/22:6) and phosphatidylethanolamine (18:0/22:6) was increased in the brain and liver of AITC-administered mice. Moreover, intragastrical injection of AITC in ovariectomized (OVX) female C57BL/6J mice dose-dependently shortened the Δlatency time determined by the Morris water maze test, indicating AITC ameliorated the cognitive function decline in these mice. Thus, the oral administration of AITC maintains GP-DHA in the liver and brain, proving to be a potential strategy for preventing cognitive decline.

Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method.

Sialidase (EC 3.2.1.18) removes sialic acid from sialoglycoconjugates. Since sialidase extracellularly applied to the rat hippocampus influences many neural functions, including synaptic plasticity and innervations of glutamatergic neurons, endogenous sialidase activities on the extracellular membrane surface could also affect neural functions. However, the distribution of sialidase activity in the brain remains unknown. To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB (FRV LB) at pH 7.3. After 1h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. These fluorescence intensities were attenuated with a sialidase-specific inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1mM). Additionally, the fluorescence intensities caused by X-Neu5Ac and FRV LB were correlated with the sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac), a classical substrate for quantitative measurement of sialidase activity, in each brain region. Therefore, staining with X-Neu5Ac and FRV LB is specific for sialidase and useful for quantitative analysis of sialidase activities. The results suggest that white matter of the rat brain has intense sialidase activity.

Plaque formation assay for human parainfluenza virus type 1.

Human parainfluenza virus type 1 (hPIV1) generally does not show visible plaques in common cell lines, including Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells, by plaque formation assays for human parainfluenza virus type 3 (hPIV3) and Sendai virus. In several conditions of the plaque formation assay, complete elimination of serum proteins in the overlay medium was necessary for visualization of hPIV1-induced plaque formation in LLC-MK(2) cells. We developed a plaque formation assay for hPIV1 isolation and titration in LLC-MK(2) cells using an initial overlay medium of bovine serum albumin-free Eagle's minimum essential medium containing agarose and acetylated trypsin for 4-6 d followed by a second overlay staining medium containing agarose and neutral red. The assay allowed both laboratory and clinical hPIV1 strains to form large plaques. The plaque reduction assay was also performed with rabbit anti-hPIV1 antibody as a general evaluation model of viral inhibitors to decrease both the plaque number and size. The results indicate that the plaque formation assay is useful for hPIV1 isolation, titration, evaluation of antiviral reagents and epidemiologic research.

Live Imaging of Virus-Infected Cells by Using a Sialidase Fluorogenic Probe.

Visualization of virus-infected cells is usually performed by immunostaining with an antiviral antibody. On the other hand, we established an easy method for fluorescence (FL) imaging of cells infected with influenza A and B viruses and some paramyxoviruses without the need for cell fixation and an antiviral antibody. These viruses and the cells they have infected express the viral surface enzyme "neuraminidase" or "hemagglutinin-neuraminidase" that shows sialidase activity. Sialidase activity is fluorescently visualized by using a sialidase fluorogenic probe developed in our previous study. The probe enables histochemical FL imaging of the virus-infected cells and is applicable to virus isolation and detection of an influenza virus resistant to antiinfluenza drugs of sialidase inhibitors.