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Aim: To establish an assay method for the determination of three kinds of biologically active components, five compounds (emodin, chrysophanol, baicalin, wogonin and berberine hydrochloride) simultaneously in Sanhuang tablets., Methods: HPLC was carried out, using a C18 column (150 mm x4. 6 mm ID, 5 microm) set at 30 degrees C, acetonitrile-0.02 mol x L(-1) acetic ammonium (adjusted pH to 3. 50 with acetic acid glacial) as mobile phase (using gradient) with flowing rate 1. 00 mL x min(-1) and detected at 270 nm., Results: The calibration curve of emodin was linear from 0.020 7 microg to 0.207 microg with r = 0.999 9, the average recovery was 99.65% with RSD 1.25%. The calibration curve of chrysophanol was linear from 0.052 microg to 0.52 microg with r = 0.999 9, and the average recovery was 100.36% with RSD 0.96%. The calibration curve of baicalin was linear from 0.250 5 microg to 2.505 microg with r =0.999 8, and the average recovery was 100.22% with RSD 1.29%. The calibration curve of wogonin was linear from 0.047 6 microg to 0.476 microg with r = 0.999 9, and the average recovery was 98.97% with RSD 1.20%. The calibration curve of berberine hydrochloride was linear from 0.053 12 microg to 0.531 2 microg with r = 0.999 5, and the average recovery was 96.02% with RSD 2.02%. The established method had also been used in the determination of the 5 compounds in 10 different batches of Sanhuang tablets., Conclusion: This method was proved to be accurate and quick, and can be used for the quality control of the preparation all-around.

To express human papillomavirus (HPV) L1 capsid protein in the recombinant strain of Shigella and study the potential of a live attenuated Shigella-based HPV prophylactic vaccine in preventing HPV infection, the icsA/virG fragment of Shigella-based prokaryotic expression plasmid pHS3199 was constructed. HPV type 16 L1 (HPV16L1) gene was inserted into plasmid pHS3199 to form the pHS3199-HPV16L1 construct, and pHS3199-HPV16L1 was electroporated into a live attenuated Shigella strain sh42. Western blotting analysis showed that HPV16L1 could be expressed stably in the recombinant strain sh42-HPV16L1. Sereny test results were negative, which showed that the sh42-HPV16L1 lost virulence. However, the attenuated recombinant strain partially maintained the invasive property as indicated by the HeLa cell infection assay. Specific IgG, IgA antibody against HPV16L1 virus-like particles (VLPs) were detected in the sera, intestinal lavage and vaginal lavage from animals immunized by sh42-HPV16L1. The number of antibody-secreting cells in the spleen and draining lymph nodes were increased significantly compared with the control group. Sera from immunized animals inhibited murine hemagglutination induced by HPV16L1 VLPs, which indicated that the candidate vaccine could stimulate an efficient immune response in guinea pig's mucosal sites. This may be an effective strategy for the development of an HPV prophylactic oral vaccine.

Objective: To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases., Methods: Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry., Results: The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc., Conclusion: The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

Objective: To study the two metal catalysts Ag/Al2O3 and Cu/Al2O3 that interdict the transmission pathway for SARS and other respiratory infectious diseases., Methods: Two metal catalysts Ag/Al2O3 and Cu/Al2O3 were pressed into wafers. One hundred microL 10(6) TCID50/mL SARS-CoV, 100 microL 10(6) PFU/mL recombinant baculovirus expressing hamster's prion protein (haPrP) protein and roughly 10(6) E. coli were slowly dropped onto the surfaces of the catalyst wafers and exposed for 5 and 20 min, respectively. After eluted from the surfaces of wafers, the infectivity of viruses and propagation of bacteria were measured. The expression of PrP protein was determined by Western blot. The morphological changes of bacteria were observed by electronic microscopy., Results: After exposure to the catalysts surfaces for 5 and 20 min, the infectivity of SARS-CoV in Vero cells and baculovirus in Sf9 cells dropped down to a very low and undetectable level, and no colony was detected using bacteria culture method. The expression of haPrP protein reduced to 21.8% in the preparation of Sf9 cells infected with recombinant baculovirus exposed for 5 min and was undetectable exposed for 20 min. Bacterial membranes seemed to be cracked and the cytoplasm seemed to be effluent from cell bodies., Conclusion: Exposures to the surfaces of Ag/Al2O3 and Cu/Al2O3 destroy the replication and propagation abilities of SARS-CoV, baculovirus and E. coli. Inactivation ability of metal catalysts needs to interact with air, utilizing oxygen molecules in air. Efficiently killing viruses and bacteria on the surfaces of the two metal catalysts has a promising potential for air-disinfection in hospitals, communities, and households.

Background: To evaluate the influence ultrasonic processing on the aggregation of PrP(Sc) in the brain extracts prepared from the scrapie-infected hamsters, and to seek for the way to prepare lower molecular PrP(Sc) polymer., Methods: The extracts of infected brains were prepared with a lysis solution, and treated with ultrasonics at various conditions during the different phases. The distribution and aggregation state of PrP(Sc) were analyzed by proteinase K treated. Western blot, and afterwards, quantitatively calculated with a commercially supplied software Image Totaltech., Results: The amount of PrP(Sc) in the supernatant of brain homogenates was 1.29-to 1.58-fold increased with appropriate sonication (15 s for 30 times). At the same conditions of ultrasound, the PrP amount in the supernatant prepared from the scrapie-infected hamster brain was significantly increased, whereas that prepared from healthy animal used as normal control showed little change. Comparative analyses of PrP(Sc) pellets prepared by high-speed centrifugation revealed that about 90% PrP(Sc) released into supernatant after ultrasound processing., Conclusion: Appropriate sonication of homogenate of scrapie-infected brain increases the extracted amount of PrP(Sc), being favorable to laboratory diagnosis. Larger molecular PrP(Sc) aggregates can be crashed by ultrasonic processing, engendering lower molecular PrP(Sc) polymers.

Objective: To expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrP(Sc) during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrP(Sc) forms., Methods: Hamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrP(Sc) were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immuno-golden EM., Results: PrP(Sc) was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrP(Sc) in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions., Conclusion: These results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrP(Sc) may indwell in brain tissues during the infection.

Objective: To understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.c.), intraperitoneal (i.p.), intragastrical (i.g.), intracardiac and intramuscular (i.m.) approaches., Methods: Hamsters were infected with crude- or fine-prepared brain extracts. The neuropathological changes, PrP(Sc) deposits, and patterns of PK-resistant PrP were analyzed by HE stain, immnunohistochemistry (IHC) assay and Western blot. Reactive gliosis and neuron loss were evaluated by glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) specific IHC., Results: The animals inoculated in i.m. and i.p. ways with crude PrP(Sc) extracts showed clinical signs at the average incubation of 69.2 +/- 2.8 and 65.5 +/- 3.9 days. Inoculation in i.c. and intracardiac ways with fine PrP(Sc) extracts (0.00035 g) caused similar, but relative long incubation of around 90 days. Only one out of eight hamsters challenged in i.g. way with low dosage (0.01 g) became ill after a much longer incubation (185 d), while all animals (4/4) with high dosage (0.04 g) developed clinical signs 105 days postinfection. The most remarkable spongiform degeneration and PrP(Sc) deposits were found in brain stem among the five challenge groups generally. The number of GFAP-positive astrocytes increased distinctly in brain stems in all infection groups, while the number of NSE-positive cells decreased significantly in cerebrum, except i.c. group. The patterns of PK-resistant PrP in brains were basically identical among the five infection routes., Conclusion: Typical TSE could be induced in hamsters by inoculating strain 263K in the five infection ways. The incubation periods in bioassays depend on infective dosage, administrating pathway and preparation of PrP(Sc). The neuropathological changes and PrP(Sc) deposits seem to be related with regions and inoculating pathways.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus, called the SARS coronavirus (SARS-CoV). Over 95% of well characterized cohorts of SARS have evidence of recent SARS-CoV infection. The genome of SARS-CoV has been sequenced and it is not related to any of the previously known human or animal coronaviruses. It is probable that SARS-CoV was an animal virus that adapted to human-human transmission in the recent past. The virus can be found in nasopharyngeal aspirate, urine and stools of SARS patients. Second generation reverse transcriptase polymerase chain reaction assays are able to detect SARS-CoV in nasopharyngeal aspirates of approximately 80% of patients with SARS within the first 3 days of illness. Seroconversion for SARS-CoV using immunofluorescence on infected cells is an excellent method of confirming the diagnosis, but antibody responses only appear around day 10 of the illness. Within the first 10 days the histological picture is that of acute phase diffuse alveolar damage (DAD) with a mixture of inflammatory infiltrate, oedema and hyaline membrane formation. Desquamation of pneumocytes is prominent and consistent. After 10 days of illness the picture changes to one of organizing DAD with increased fibrosis, squamous metaplasia and multinucleated giant cells. The role of cytokines in the pathogenesis of SARS is still unclear.

Objective: The causal agent for SARS is considered as a novel coronavirus that has never been described both in human and animals previously. The stability of SARS coronavirus in human specimens and in environments was studied., Methods: Using a SARS coronavirus strain CoV-P9, which was isolated from pharyngeal swab of a probable SARS case in Beijing, its stability in mimic human specimens and in mimic environment including surfaces of commonly used materials or in household conditions, as well as its resistance to temperature and UV irradiation were analyzed. A total of 10(6) TCID50 viruses were placed in each tested condition, and changes of the viral infectivity in samples after treatments were measured by evaluating cytopathic effect (CPE) in cell line Vero-E6 at 48 h after infection., Results: The results showed that SARS coronavirus in the testing condition could survive in serum, 1:20 diluted sputum and feces for at least 96 h, whereas it could remain alive in urine for at least 72 h with a low level of infectivity. The survival abilities on the surfaces of eight different materials and in water were quite comparable, revealing reduction of infectivity after 72 to 96 h exposure. Viruses stayed stable at 4 degrees C, at room temperature (20 degrees C) and at 37 degrees C for at least 2 h without remarkable change in the infectious ability in cells, but were converted to be non-infectious after 90-, 60- and 30-min exposure at 56 degrees C, at 67 degrees C and at 75 degrees C, respectively. Irradiation of UV for 60 min on the virus in culture medium resulted in the destruction of viral infectivity at an undetectable level., Conclusion: The survival ability of SARS coronavirus in human specimens and in environments seems to be relatively strong. Heating and UV irradiation can efficiently eliminate the viral infectivity.

Objective: To obtain monoclonal antibodies (McAbs) which can be widely used to detect mammalian prions (PrP) and to develop diagnostic tests for screening transmissile spongiform encephalopathies (TSE) as well as for studying pathogenesis of prion-related diseases., Methods: BALB/c mice were immunized separately with bovine PrP peptide 29-48 (BoP1) and 89-108 (BoP2) coupled to keyhole limpt hemocyan. Two hybridoma cell lines secreting monoclonal antibodies against these peptides were established by cell fusion and 2 to 3 rounds of cell cloning. The reactions of the McAbs to the recombinant bovine (Bo)PrP(25-242), human (Hu)PrP(23-231) and hamster (Ha) PrP (23?231) were tested separately by Western blotting., Results: Through cell fusion, two hybridoma cell lines secreting McAbs against BoP1 and BoP2, designated D11 and D8 accordingly, were identified by ELISA and cell cloning. The McAbs produced by these cell lines reacted well with the recombinant PrP proteins; (Bo) PrP (25-242), (Hu) PrP (23-231), and (Ha) PrP (23-231), respectively., Conclusions: Two McAbs reacting with bovine, human and hamster PrPs were successfully generated, they are potential to be used to detect PrPs in mammals and to study the mechanism of pathogenesis of TSE.

Objective: To explore the causative agents of the atypical pneumonia (also SARS) occurred recently in some regions of our country., Method: Organ samples of 7 dead cases of SARS were collected from Guangdong, Shanxi, Sichuan Provinces and Beijing for electron microscopic examination. 293 cell line was inoculated with the materials derived from the lungs to isolate causative agent(s). The agents in the organs and cell cultures were revealed by immunoassay., Results: Both Chlamydia-like and coronavirus-like particles were found in EM. Inclusion bodies containing elementary bodies, reticulate antibodies and intermediate bodies of Chlamydia-like agent were visualized in multiple organs from the 7 dead cases, including lungs (7 cases), spleens (2 cases), livers (2 cases), kidneys (3 cases) and lymph nodes (1 cases), by ultrathin section electron microscopy (EM). In some few sections, coronavirus-like particles were concurrently seen. A coronavirus RNA- polymerase segment (440 bp) was amplified from the lung tissues of two cases of the SARS. After inoculated with materials from the lung samples, the similar Chlamydia-like particles were also found in the inoculated 293 cells. Since the Chlamydia-like agents visualized in both organs and cell cultures could not react with the genus specific antibodies against Chlamydia and monoclonal antibodies against C. pneumoniae and C. psittaci, the results might well be suggestive of a novel Chlamydia-like agent., Conclusion: Since the novel Chlamydia-like agent was found co-existing with a coronavirus-like agent in the dead cases of SARS, it looks most likely that both the agents play some roles in the disease. At the present time, however, one can hardly determining how did these agents interact each other synergetically, or one follows another, need further study.

Background: To develop HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines., Methods: The nucleotides within HPV 16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mega primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV 16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV 16 L1 and E6 specific monoclonal antibodies., Results: ELISA showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were greater than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells., Conclusions: Successful constructions of prophylactic and therapeutic DNA vaccine plasmids may be useful for future animal experiment and clinical trial.

Objective: To clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis., Methods: Using PCR, the prtH was amplified and cloned into pGEM-T vector. To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe., Results: Recombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay. Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns. Strain 47A-1 seemed even not to contain prtH gene., Conclusions: Different prtH gene sequences exist in different P.g strains. This polymorphism may indicate various potential virulent effects during the infection and pathogenesis. Established PCR protocol is sensitive for identification of prtH gene.