An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (α and β), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (α and β) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (α and β) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis.

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus was compared with viral culture in embryonated chicken eggs. Virus was isolated from blood or anal swabs of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). Viral nucleic acid was detected in blood samples by NASBA/ECL immediately prior to death, whilst nucleic acid extracted from anal swabs was detected from the day following artificial infection until death. Thus, blood and/or anal swabs are a suitable source of material for the detection of avian influenza in dead birds, but anal swabs are more suitable for detection of viral genetic material in live birds. Dilution of a known viral standard was used to determine the limit of sensitivity for both NASBA/ECL and egg culture detection methods. The NASBA/ ECL method was equivalent in sensitivity to egg culture. The NASBA/ECL results agreed with egg culture data in 71/94 (75.5%) tissue samples obtained from artificially infected birds.

The Six Cities Study by Dockery et al.1 has captured renewed attention (Jan. 8 issue).2,3 Claiming to identify an increased mortality rate in Steubenville, Ohio, as compared with the rate in Portage, Wisconsin, the authors posited that air pollution in Steubenville, an Ohio River Valley mining and steel-making city, was responsible for its increased death rate. That interpretation is not permissible, inasmuch as the socioeconomic and ethnic makeups of the populations of Portage and Steubenville are markedly different. Compounding that invidious comparison, the calculated difference in mortality rates is seen to be only a hair’s breadth within the bounds of statistical significance. Indeed, as shown in Table 2 of the report,1 there was no difference in mortality between women in Portage and women in Steubenville, who breathed the same air as men in that city. Steinbrook3 quotes David M. Michaels, formerlyformerly of the Clinton administration, who suggests that critics of clean-air studies are “hired guns working for dirty companies.” Joel Schwartz, later associated with the clean-air group at Harvard, reportedly dismissed critics of clean-air studies as “industry thugs.”4 Is this the language of collegial scientific discourse?

In early 2003, a novel severe acute respiratory syndrome (SARS) coronavirus (CoV) [1] spread around the world; ultimately, more than 8000 patients in 32 countries contracted SARS, many of whom died. Although gold standard methods, such as viral culture, can help diagnose SARS, these methods are by no means as efficient and rapid as PCR-based diagnostic tests. The speed and sensitivity of molecular diagnostic tests for SARS is often considerably greater than than that of serological and viral culture methods [2]. Our reported enhanced real-timePCR (ERT) method [3, 4] is 100-fold more sensitive than conventional real-timePCR. The higher sensitivity of this method may reveal potential SARS CoV carriers who have SARS CoV levels that are undetectable by other methods, and the sensitivity of the ERT method may be particularly important for ensuring that patients who have had SARS are not infectious before discharge from the hospital [5]. In collaboration with Princess Margaret Hospital (PMH; Hong Kong), samples obtained from 3 patients during recovery after SARS were analyzed (table 1). Six to nine weeks after the onset of infection, SARS CoV could still be detected by ERT in certain samples (table 1), indicating that, although clinical signs and symptoms had subsided and a host immune response had been mounted, viral clearance was not complete. Patient 1 was transferred on 17 June 2003 to the Wong Tai Sin Hospital (WTSH; Hong Kong), which was converted into a specialized center for convalescent care of patients with SARS during the epidemic, but he was returned to PMHbecause of recurrent pneumothorax, indicated by chest radiography on 18 June. The ERT method clearly demonstrated the presence of SARS CoV in all samples obtained from the patient on 16 June (table 1), which was 1 day before his transfer to WTSH. The possible relapse of infection in patient 1 after his transfer to another hospital indeed raises the question of how patients with SARS who have PCR results negative for SARS CoV should be handled [5]. Standardization of clinical criteria and PCR-based methods should be emphasized to ensure accurate diagnosis of SARS after hospital admission and prior to hospital discharge. More studies will be necessary to determine the infectivity status of patients who have ERT results positive for SARS CoV. The data suggest that medical professionals should verify whether residual viral particles present in recovering patients remain infectious and whether they may constitute the source of possible future outbreaks of infection. Because SARS is a newly emerging disease that causes serious consequences, many countries have formulated contingency plans for possible future SARS outbreaks. One of the containment activities currently undertaken by theWorld Health Organization to prevent SARS from repeatedly becoming a widely established threat is to develop a robust and reliable diagnostic test [6], which will probably rely on PCR-based technology. Use of a highly sensitive method, such as the ERT method [3, 4] and a similar method that was reported recently [7], will be the first step toward more accurate screening of suspected SARS carriers and will minimize the occurrence of false-negative cases. Patients with false-positive cases can always be quarantined while awaiting further reconfirmation of infection. But patients with false-negative cases could be discharged into the community and pose a dangerous SARS threat to the public [8]. Therefore, stringent clinical criteria and use of the ERT method might effectively monitor patients recovering after SARS.

An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (α and β), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (α and β) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (α and β) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis.

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.

The 14-3-3 protein family comprises critical regulatory molecules involved in signaling during cell division, proliferation, and apoptosis. Despite extensive study, the functions of the 14-3-3 proteins in brain remain unclear. 14-3-3γ, a subtype of the 14-3-3 family of proteins, was thought to be brain-and neuron-specific. Using RNA arbitrarily primed PCR, we identified an upregulated cDNA fragment of the 14-3-3γ gene in primary cultures of astrocytes. Using Northern blot analysis, we confirmed this fragment was brain-specific. In cultures of astrocytes, 14-3-3γgenes and proteins were differentially expressed at different ages and the proteins were distributed only in the cytoplasm. These results indicated that 14-3-3 was not neuron-specific but also expressed in astrocytes. The function of this protein in brain is unclear. Northern and Western blot analyses demonstrated that 14-3-3γmRNA and protein were upregulated in cultured astrocytes in an anaerobic chamber-induced ischemia model. The induction of 14-3-3γproteins was neither suppressed by an MAP kinase inhibitor (U0126) nor a PI-3 kinase inhibitor (LY294002). These data indicated that induction of 14-3-3γmight not involve PI-3 and MAP kinase-dependent pathways. Using coimmunoprecipitation, we demonstrated that endogenous 14-3-3γbound to c-Raf-1 and p-Raf 259. As Raf is one of the critical serine/threonine kinases controlling cell growth, differentiation, and death, the binding of 14-3-3γto Raf indicates the critical role of this protein in ischemiainduced apoptosis and the changes in signal transduction in astrocytes in culture. GLIA 42: 315-324, 2003.

The nucleotide sequence of the VP1 coding region of foot-and-mouth disease virus (FMDV) strain HKN/2002, isolated from a disease outbreak occurring in Hong Kong in February 2002, was determined and compared with the sequences of other FMDVs. The VP1 coding region was 639 nucleotides in length and encoded a protein of 213 amino acid residues. Comparison of the VP1 nucleotide sequence with those of other isolates indicated that HKN/2002 belonged to serotype O. A VP1-based sequence similarity tree of several South-east Asian FMDV-O isolates showed that HKN/2002 was most closely related to FMDV isolates found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Comparison of the amino acid sequence of the major immunogenic region of HKN/2002 with that of the serotype O vaccine strain, O1/Manisa/Turkey/69, reveals significant similarity, indicating that current serotype O vaccines may offer some degree of protection against HKN/2002.