Yin-Wan Wendy Fung

Nucleic acid sequence-based amplification methods to detect avian influenza virus

Abstract Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1–H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl’s eggs.

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

Abstract An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 102-fold higher than the standard real-time PCR assay and 107-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

Presence of Neuroglobin in Cultured Astrocytes

Neuroglobin (Ngb), a recently discovered intracellular respiratory globin in neurons, may play a crucial role in oxygen homeostasis in the brain. We report preliminary findings indicating the presence of functional neuroglobin in primary cultures of cerebral cortical astrocytes. Reverse transcription real‐time polymerase chain reaction (RRT–PCR) and immunostaining confirmed such presence in cultured astrocytes isolated from newborn mouse brain. Ngb antisense treatment increased apoptosis in ischemic astrocytes. The discovery of Ngb in astrocytes may provide some insight into how oxygen homeostasis is regulated in the brain.

Association of 14-3-3γ and Phosphorylated Bad Attenuates Injury in Ischemic Astrocytes

Our recent findings indicate an induced upregulation of 14-3-3γ mRNA and protein in ischemic cortical astrocytes. Despite being brain-specific, the functional role of 14-3-3γ in the brain still remains largely unknown. In this study, we show that among all the 14-3-3 isoforms, only the γ isoform is inducible under ischemia in astrocytes. Furthermore, this upregulation of 14-3-3γ may play a specific protective role in astrocytes under ischemia. Overexpression experiments and antisense treatment show that an elevation of 14-3-3γ protein in astrocytes promotes survival, while a decrease in 14-3-3γ enhances apoptosis in astrocytes under ischemia. Under ischemia, endogenous 14-3-3γ binds p-Bad, thus preventing Bad from entering mitochondria to initiate apoptosis. Therefore, 14-3-3γ is selectively induced during ischemia to protect astrocytes from apoptosis through p-Bad-related signaling.

Selective regulation of 14-3-3η in primary culture of cerebral cortical neurons and astrocytes during development

The 14‐3‐3 proteins exist predominantly in the brain and may play regulatory roles in cellular processes of growth, differentiation, survival, and apoptosis. The biological functions, however, of the various 14‐3‐3 isoforms (β, ϵ, η, γ, and ζ) in the brain remain unclear. We have reported previously upregulation of 14‐3‐3γ in ischemic astrocytes. In the present study, we report selective regulation of 14‐3‐3η in cultured cerebral cortical neurons and astrocytes during in vitro development. In cultured neurons, gene expression levels of 14‐3‐3η increase with culture age (0–10 days). Brain‐derived neurotrophic factor and neurotrophin‐3 upregulate 14‐3‐3η gene expression. In cultured astrocytes, 14‐3‐3η is downregulated with culture age (1–5 weeks). The gene expression level of 14‐3‐3η is not affected by scratch injury in astrocytes or by ischemia in neurons. These data suggest a possible role of 14‐3‐3η in growth and differentiation of neurons and astrocytes, indicating an intricate mechanism governing coordinated and well‐controlled developmental events in the brain to ensure normal neural functions.

Inactivation of Bad by Site-Specific Phosphorylation: The Checkpoint for Ischemic Astrocytes to Initiate or Resist Apoptosis

Bcl‐2‐associated death protein (Bad), a member of the Bcl family, directs astrocytes in primary cultures to enter or resist apoptosis during ischemia in vitro. Under ischemia, Bad was the only Bcl family member whose expression was upregulated significantly during the early stages of an ischemic insult. Increased endogenous Bad was translocated from the cytoplasm to mitochondria to induce apoptosis in astrocytes. Concurrently, ischemia also induced Bad phosphorylation specifically on Ser112 to promote survival. This site‐specific phosphorylation of Bad was mediated by an early activation of the mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase (MAPK/ERK) intracellular signaling pathway. This study demonstrates that ischemia‐induced Bad plays a dual role in determining whether astrocytes enter or resist apoptosis after an ischemic insult.

Boosting the sensitivity of real-time polymerase-chain-reaction testing for SARS.

To the Editor: In view of recent concern about the recurrence of severe acute respiratory syndrome (SARS) in Guangdong, China, we would like to highlight the tremendous importance of sensitivity in...

Astrocytes in Migration.

Cell migration is a fundamental phenomenon that underlies tissue morphogenesis, wound healing, immune response, and cancer metastasis. Great progresses have been made in research methodologies, with cell migration identified as a highly orchestrated process. Brain is considered the most complex organ in the human body, containing many types of neural cells with astrocytes playing crucial roles in monitoring normal functions of the central nervous system. Astrocytes are mostly quiescent under normal physiological conditions in the adult brain but become migratory after injury. Under most known pathological conditions in the brain, spinal cord and retina, astrocytes are activated and become hypertrophic, hyperplastic, and up-regulating GFAP based on the grades of severity. These three observations are the hallmark in glia scar formation—astrogliosis. The reactivation process is initiated with structural changes involving cell process migration and ended with cell migration. Detailed mechanisms in astrocyte migration have not been studied extensively and remain largely unknown. Here, we therefore attempt to review the mechanisms in migration of astrocytes.

RNA-dependent RNA polymerase gene sequence from foot-and-mouth disease virus in Hong Kong

A foot-and-mouth disease virus (FMDV, HKN/2002) was isolated in Hong Kong in 2002. The nucleotide sequence of the 3D(pol) gene encoding the viral RNA-dependent RNA polymerase was determined and compared with that of the same gene from other FMDVs. The 3D(pol) gene was 1410 nucleotides in length encoding a protein of 470 amino acid residues. Sequence comparisons indicated that HKN/2002 belonged to serotype O. An evolutionary tree based on the 3D(pol) sequences of 20 FMDV isolates revealed that the nucleotide sequence of the HKN/2002 3D(pol) gene was most similar to those of isolates found in Taiwan in 1997, suggesting that they share a common ancestor. The amino acid sequence of the HKN/2002 3D(pol) gene was determined and aligned with those of representative isolates from seven other Picornaviridae genera. Eight highly conserved regions were detected, indicating a conserved functional relevance for these motifs. Alignment of 20 FMDV 3D(pol) amino acid sequences revealed a hypermutation region near the N-terminus that may help the virus evade host immune systems.

Identification of a mouse synaptic glycoprotein gene in cultured neurons.

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0–2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0–2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.