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.

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.

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.

Enhancement of elastin expression by transdermal administration of sialidase isozyme Neu2.

Reduction of elastin in the skin causes various skin diseases as well as wrinkles and sagging with aging. Sialidase is a hydrolase that cleaves a sialic acid residue from sialoglycoconjugate. Cleavage of sialic acid from microfibrils by the sialidase isozyme Neu1 facilitates elastic fiber assembly. In the present study, we showed that a lower layer of the dermis and muscle showed relatively intense sialidase activity. The sialidase activity in the skin decreased with aging. Choline and geranate (CAGE), one of the ionic liquids, can deliver the sialidase subcutaneously while maintaining the enzymatic activity. The elastin level in the dermis was increased by applying sialidase from Arthrobacter ureafaciens (AUSA) with CAGE on the skin for 5 days in rats and senescence-accelerated mice prone 1 and 8. Sialidase activity in the dermis was considered to be mainly due to Neu2 based on the expression level of sialidase isozyme mRNA. Transdermal administration of Neu2 with CAGE also increased the level of elastin in the dermis. Therefore, not only Neu1 but also Neu2 would be involved in elastic fiber assembly. Transdermal administration of sialidase is expected to be useful for improvement of wrinkles and skin disorders due to the loss of elastic fibers.

The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid is a glucose-dependent potentiator of insulin secretion.

Sialidase cleaves sialic acid residues from a sialoglycoconjugate: oligosaccharides, glycolipids and glycoproteins that contain sialic acid. Histochemical imaging of the mouse pancreas using a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe used to assess sialidase activity, showed that pancreatic islets have intense sialidase activity. The sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) remarkably enhances glutamate release from hippocampal neurons. Since there are many similar processes between synaptic vesicle exocytosis and secretory granule exocytosis, we investigated the effect of DANA on insulin release from ß-cells. Insulin release was induced in INS-1D cells by treatment with 8.3 mM glucose, and the release was enhanced by treatment with DANA. In a mouse intraperitoneal glucose tolerance test, the increase in serum insulin levels was enhanced by intravenous injection with DANA. However, under fasting conditions, insulin release was not enhanced by treatment with DANA. Calcium oscillations induced by 8.3 mM glucose treatment of INS-1D cells were not affected by DANA. Blood insulin levels in sialidase isozyme Neu3-deficient mice were significantly higher than those in WT mice under ad libitum feeding conditions, but the levels were not different under fasting conditions. These results indicate that DANA is a glucose-dependent potentiator of insulin secretion. The sialidase inhibitor may be useful for anti-diabetic treatment with a low risk of hypoglycemia.

Fluorogenic Probes for Accurate in Situ Imaging of Viral and Mammalian Sialidases.

Sialidases are widely distributed in nature and are involved in many physiological and pathological processes. Sialidases are expressed and work in various tissues and organelles. Clarification of the localization of sialidases is very helpful as a way to understand their functions. We previously developed a novel fluorogenic probe for sialidases, BTP3-Neu5Ac, that visualized the localization of sialidase activity in live cells and tissues by precipitating the hydrophobic fluorescent compound; however, for the purpose of accurate fluorescence imaging of sialidase-expressing cells or the distribution of intracellular sialidase activity, BTP3-Neu5Ac was inadequate in imaging performance. We report the design and development of a sialidase imaging probe that improves the sensitivity and accuracy of in situ fluorescence imaging performance as well as increases the hydrophobicity by attaching linear unsaturated hydrocarbon chains into the hydrophobic fluorescent compound of BTP3-Neu5Ac. The newly developed probe showed low diffusivity and high brightness for fluorescence imaging, and it enabled sensitive and highly accurate imaging of viral sialidase in virus-infected cells and sialidase-expressing cells as well as mammalian sialidase in the rat brain. The probe also enabled the fluorescence imaging of intracellular viral sialidase in live-virus-infected cells. The newly developed probe is expected to be a useful tool that will contribute to the progress of research on sialidases in various fields such as research on viruses and brains.

Application of Novel Sialoglyco Particulates Enhances the Detection Sensitivity of the Equine Influenza Virus by Real-Time Reverse Transcriptase Polymerase Chain Reaction.

Sialoglyco particulates carrying an N-glycolylneuraminyl-α-(2 → 3)-N-acetyllactosamine (Neu5Gcα2,3LacNAc) residue that displays a high level of affinity for the equine influenza virus (EIV) were generated using sialoglycopolypeptide and hexyl-containing hybrid silica particulates. The particulates were spherical with a diameter of approximately 950 nm and found to have good dispersibility in aqueous solution. Interaction between the sialoglyco particulates and the EIV was investigated by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) of the EIV genome captured on the particulates. The number of EIV-specific genes detected by rRT-PCR on a nasal swab obtained from infected horses clearly increased when the sample was treated with sialoglyco particulates. Our results show these novel sialoglyco particulates can be used as a highly sensitive tool for detecting low levels of EIV that were previously undetectable in the early or late stage of infection.

An easy, rapid, and sensitive method for detection of drug-resistant influenza virus by using a sialidase fluorescent imaging probe, BTP3-Neu5Ac.

Immunochromatographic kits and RT-PCR are widely used as diagnostic tools for influenza detection in clinical and hygiene fields. Immunochromatographic kits are useful for differential typing of influenza A and influenza B but cannot show if the detected virus strains have acquired drug resistance against neuraminidase inhibitors that target sialidase activity of viral neuraminidase. Although RT-PCR enables determination of drug-resistant mutants, its efficacy is limited to viruses carrying a known substitution in their neuraminidase genome sequence. In the present study, an easy, rapid and sensitive method for detection of drug-resistant influenza viruses regardless of major antigenic changes or genomic mutations was developed. By using the method in combination with virus-concentrated membranes in centrifugal filter units and a sialidase imaging probe, 2-(benzothiazol-2-yl)-4-bromophenyl-N-acetylneuraminic acid (BTP3-Neu5Ac), sialidase activity of influenza neuraminidase was visualized on membranes by the green fluorescence of produced hydrophobic BTP3 under UV irradiation with a handheld UV flashlight. Fluorescence images in the presence or absence of neuraminidase inhibitors clearly discriminated drug-resistant influenza viruses from drug-sensitive ones. The assay can be done within 15 min. The detection sensitivity was shown to be equal to or higher than the sensitivities of commercial immunochromatographic kits. The assay will be a powerful tool for screening and monitoring of emerging drug-resistant influenza viruses and would help clinicians decide effective antiviral treatment strategies when such mutants have become prevalent.

Down-regulation of glutamate release from hippocampal neurons by sialidase.

Sialidase, which removes sialic acid residues in sialylglycoconjugates, is essential for hippocampal memory and synaptic plasticity. Enzyme activity of sialidase is rapidly increased in response to neural excitation. Because sialic acid bound to gangliosides such as the tetra-sialoganglioside GQ1b is crucial for calcium signalling and neurotransmitter release, neural activity-dependent removal of sialic acid may affect hippocampal neurotransmission. In the present study, we found that 2-deoxy-2, 3-didehydro-D-N-acetylneuraminic acid (DANA), a sialidase inhibitor, increased expression of ganglioside GQ1b/GT1a in hippocampal acute slices. Extracellular glutamate level in the rat hippocampus measured by using in vivo microdialysis was increased by the sialidase inhibitor 2, 3-dehydro-2-deoxy-N-glycolylneuraminic acid as well as DANA. Synaptic vesicle exocytosis and intracellular Ca2+ increase evoked by high-K+ were also enhanced by DANA in primary cultured hippocampal neurons. Expression of GQ1b/GT1a was rapidly decreased by depolarization with high-K+, suggesting that the increase in sialidase activity by neural excitation is sufficient for cleavage of sialic acid. Our findings indicate that sialidase down-regulates glutamate release from hippocampal neurons via Ca2+ signalling modulation. Neural activity-dependent desialylation by sialidase may be a negative-feedback factor against presynaptic activity.

A novel, simplified strategy of relative quantification N-glycan: Quantitative glycomics using electrospray ionization mass spectrometry through the stable isotopic labeling by transglycosylation reaction of mutant enzyme Endo-M-N175Q.

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8=0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R2=0.9999; d8/d0, R2=0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC-MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.

Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity.

Aeromonas sobria serine protease (ASP) is an extracellular serine protease secreted by the organism. Here, we identified the amino acid residue of ASP that contributes to substrate specificity by using both synthetic peptides and biological protein components. The results showed that the arginine residue at position 566 (Arg-566) of ASP, which is located in the extra occluding region of ASP close to an entrance of the catalytic cavity, is involved in the substrate specificity. A substitutional point mutation of the Arg-566 residue of ASP to Ala residue (ASP[R566A]) caused a decrease of the proteolytic efficiency for a certain substrate. In addition, ASP lost the ability to recognize the primary substrate by such a point mutation, and ASP[R566A] reacted to a wide range of synthetic substrates. It is likely that Arg-566 causes an interaction with the amino acid residue at position P3 of the substrate, which is the third amino acid residue upstream from the cleavage site. Another study using ORF2 protein, a chaperone protein of ASP, further suggested that Arg-566 could also play an important role in interaction with ORF2. We therefore conclude that the Arg-566 residue of ASP is likely responsible for the selection of substrates.

Chemoenzymatic synthesis and characterization of N-glycolylneuraminic acid-carrying sialoglycopolypeptides as effective inhibitors against equine influenza virus hemagglutination.

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 â†’ 3)- and α(2 â†’ 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a-c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.

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.

Role of Sialidase in Long-Term Potentiation at Mossy Fiber-CA3 Synapses and Hippocampus-Dependent Spatial Memory.

Sialic acid bound to glycans in glycolipids and glycoproteins is essential for synaptic plasticity and memory. Sialidase (EC 3.2.1.18), which has 4 isozymes including Neu1, Neu2, Neu3 and Neu4, regulates the sialylation level of glycans by removing sialic acid from sialylglycoconjugate. In the present study, we investigated the distribution of sialidase activity in rat hippocampus and the role of sialidase in hippocampal memory processing. We previously developed a highly sensitive histochemical imaging probe for sialidase activity, BTP3-Neu5Ac. BTP3-Neu5Ac was cleaved efficiently by rat Neu2 and Neu4 at pH 7.3 and by Neu1 and Neu3 at pH 4.6. When a rat hippocampal acute slice was stained with BTP3-Neu5Ac at pH 7.3, mossy fiber terminal fields showed relatively intense sialidase activity. Thus, the role of sialidase in the synaptic plasticity was investigated at mossy fiber terminal fields. The long-term potentiation (LTP) at mossy fiber-CA3 pyramidal cell synapses was impaired by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), a sialidase inhibitor. DANA also failed to decrease paired-pulse facilitation after LTP induction. We also investigated the role of sialidase in hippocampus-dependent spatial memory by using the Morris water maze. The escape latency time to reach the platform was prolonged by DANA injection into the hippocampal CA3 region or by knockdown of Neu4 without affecting motility. The results show that the regulation of sialyl signaling by Neu4 is involved in hippocampal memory processing.

High-Efficiency Capture of Drug Resistant-Influenza Virus by Live Imaging of Sialidase Activity.

Influenza A and B viruses possess a neuraminidase protein that shows sialidase activity. Influenza virus-specific neuraminidase inhibitors (NAIs) are commonly used for clinical treatment of influenza. However, some influenza A and B viruses that are resistant to NAIs have emerged in nature. NAI-resistant viruses have been monitored in public hygiene surveys and the mechanism underlying the resistance has been studied. Here, we describe a new assay for selective detection and isolation of an NAI-resistant virus in a speedy and easy manner by live fluorescence imaging of viral sialidase activity, which we previously developed, in order to achieve high-efficiency capture of an NAI-resistant virus. An NAI-resistant virus maintains sialidase activity even at a concentration of NAI that leads to complete deactivation of the virus. Infected cells and focuses (infected cell populations) of an oseltamivir-resistant virus were selectively visualized by live fluorescence sialidase imaging in the presence of oseltamivir, resulting in high-efficiency isolation of the resistant viruses. The use of a combination of other NAIs (zanamivir, peramivir, and laninamivir) in the imaging showed that the oseltamivir-resistant virus isolated in 2008 was sensitive to zanamivir and laninamivir but resistant to peramivir. Fluorescence imaging in the presence of zanamivir also succeeded in selective live-cell visualization of cells that expressed zanamivir-resistant NA. Fluorescence imaging of NAI-resistant sialidase activity will be a powerful method for study of the NAI resistance mechanism, for public monitoring of NAI-resistant viruses, and for development of a new NAI that shows an effect on various NAI-resistant mutations.

Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

Preventive Effect of 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) and Zinc, Components of the Pacific Oyster Crassostrea gigas, on Glutamatergic Neuron Activity in the Hippocampus.

The effects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), and zinc--both components of the Pacific oyster Crassostrea gigas--were examined by glutamatergic neuron activity in rats in an in vivo microdialysis experiment and an in vitro brain slice experiment. The basal concentration of extracellular glutamate in the hippocampus was decreased under hippocampal perfusion with DHMBA (1 mmol l(-1)) or ZnCl2 (µmol l(-1)), indicating that DHMBA and Zn(2+) suppress glutamatergic neuron activity under basal (static) conditions. To assess the preventive effect of DHMBA and Zn(2+) on glutamate release from neuron terminals, brain slices were pretreated with DHMBA (1 mmol l(-1)) or ZnCl2 (100 nmol l(-1)) for 1 h, then stimulated with high K(+). A high, K(+)-induced increase in extracellular Zn(2+) level, an index of glutamate release, was suppressed with pretreatment with DHMBA or zinc. A high, K(+)-induced increase in intracellular Ca(2+) level was also suppressed with pretreatment with DHMBA or Zn(2+). These results suggest that DHMBA and Zn(2+), previously taken up in the hippocampal cells, suppress high, K(+)-induced glutamate release in the hippocampus, probably via presynaptic suppression of intracellular Ca(2+) signaling. It is likely that Zn(2+) and DHMBA play a preventive role in suppressing excess glutamatergic neuron activity in rats and mice.

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.

Preferential Accumulation of 14C-N-Glycolylneuraminic Acid over 14C-N-Acetylneuraminic Acid in the Rat Brain after Tail Vein Injection.

The two main molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, 14C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of 14C-Neu5Gc was excreted in urine, a small amount of 14C-Neu5Gc was detected in the brain. Brain autoradiography indicated that 14C-Neu5Gc was accumulated predominantly in the hippocampus. 14C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of 14C-Neu5Gc after intracerebroventricular infusion was slower than that of 14C-Neu5Ac in the brain and hippocampus. The results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain more preferentially than does Neu5Ac.