Novel daidzein analogs and their in vitro anti-influenza activities.

A series of novel isoflavonoids were synthesized based on structural modifications of daidzein, an active ingredient of traditional Chinese medicine (TCM) and evaluated for their anti-influenza activity, in vitro, against H1N1 Tamiflu-resistant (H1N1 TR) virus in the MDCK cell line. Among them, 4-oxo-4H-1-benzopyran-8-carbaldehydes 11a-11g were most promising, and they demonstrated better activities and selectivities comparable to those the reference ribarivin, a nucleoside antiviral agent. 3-(4-Bromophenyl)-7-hydroxy-4-oxo-4H-1-benzopyran-8-carboxaldehyde (11c) displayed the best inhibitory activity (EC50 , 29.0 µM) and selectivity index (SI>10.3). Analysis of the structureactivity relationships (SAR) indicated that both the non-naturally-occurring Br-substituted B-ring and appropriate CHO and OH groups on the A-ring might be critical for the activity and selectivity against H1N1 TR influenza viruses.

Anti-cancer agents derived from solid-state fermented Antrodia camphorata mycelium.

Three new ubiquinone derivatives, antrocamol LT1, antrocamol LT2, and antrocamol LT3, along with two known compounds, were isolated from Antrodia camphorata (Polyporaceae) mycelium. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analyses. These ubiquinones exhibited selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 1.79µΜ.

Cytotoxic effect of triterpenoids from the root bark of Hibiscus syriacus.

In this study, 4 new triterpenoids-3ß- acetoxy-olean-11-en,28,13ß-olide (1), 3ß- acetoxy-11α,12α-epoxy-olean-28,13ß-olide (2), 19α-epi-betulin (3), and 20, 28-epoxy-17ß,19ß-lupan-3ß-ol (4)-and 12 known compounds, were isolated from the root bark of Hibiscus syriacus L. by using acetone extraction. Their structures were characterized by extensive spectroscopic analysis. To investigate cytotoxicity, A549 human lung cancer cells were exposed to the extract and the compounds identified from it. Significantly reduced cell viability was observed with betulin-3-caffeate (12) (IC50, 4.3 µM). The results of this study indicate that betulin-3-caffeate (12) identified from H. syriacus L. may warrant further investigation for potential as anticancer therapies.

Synthesis and anti-influenza activities of novel baicalein analogs.

A series of novel flavones derivatives were synthesized based on modification of the active ingredients of a traditional Chinese medicine Scutellaria baicalensis GEORGI and screened for anti-influenza activity. The synthetic baicalein (flavone) analogs, especially with the B-rings substituted with bromine atoms, were much more potent than oseltamivir or ribavirin against H1N1 Tamiflu-resistant (H1N1 TR) virus and usually with more favorable selectivity. The most promising were 5b, 5c, 6b and 6c, all displaying an 50% effective concentration (EC50) at around 4.0-4.5 µM, and a selective index (SI=50% cytotoxic concentration (CC50)/EC50)>70. For seasonal H3N2-infected influenza virus, both 5a and 5b with SI >17.3 indicated superior to ribavirin. The flavonoids having both not-naturally-occurring bromo-substituted B-rings and appropriate hydroxyls positioning on the A-rings might be critical in determining the activity and selectivity against H1N1-Tamiflu-resistant infected influenza viruses.

Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: implications for the diagnosis of coronavirus infection.

Previous studies have reported that a prokaryotic-expressed recombinant nucleocapsid protein (NP) is a suitable reagent for the epidemiological screening of coronavirus infection. In this study, soluble recombinant human coronavirus OC43 (HCoV-OC43) NP was produced to examine the antigenicity of the HCoV-OC43 NP of betacoronavirus. Using the purified recombinant NP as an antigen, a polyclonal antibody from rabbit serum with specificity for HCoV-OC43 NP was generated; this antibody reacts specifically with HCoV-OC43 NP and does not cross-react with other human CoV NPs (including those of SARS-CoV and HCoV-229E) by Western blot. Sera from 26 young adults, 17 middle-aged and elderly patients with respiratory infection, and 15 cord blood samples were also tested. Strong reactivity to the NPs of HCoV-OC43 was observed in 96%, 82%, and 93% of the serum samples from the young adults, respiratory patients, and cord blood samples, respectively. To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum, the antigenicities of three protein fragments, including the N-terminal domain (aa 1-173), the central-linker region (aa 174-300), and the C-terminal domain (aa 301-448), were evaluated by Western blot. The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central-linker region and the C-terminal domain than to the N-terminal domain. Three different patterns for the immunoreactivities of the three structural regions of HCoV-OC43 NP were observed in human serum, suggesting variability in the immune responses that occur during HCoV-OC43 infection in humans. The central-linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed. The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection.

Toluene diisocyanate (TDI) induces calcium elevation and interleukine-4 (IL-4) release - early responses upon TDI stimulation.

Occupational exposure to toluene diisocyanats (TDI) may cause asthma. In asthma patients, the allergic syndromes correlate cytokine production with the elevation in cytosolic calcium concentration [Ca(2+)](c) of lymphocytes in airway. We previously found TDI induces calcium signaling in neuronal cells. TDI mainly gets into human body via inhalation; therefore this study investigated the possibility of TDI inducing the changes in [Ca(2+)](c) in airway. We used human lung epithelial cell line H1355, human T-cell line Jurkat, and human neuroblastoma SH-SY5Y cells to present the kinds of cells existing in airway. The changes of [Ca(2+)](c) were measured by Fura-2 fluorescent dye. Results show that TDI induced an elevation in [Ca(2+)](c )in those cell lines and two primary isolated cells, bovine adrenal chromaffin cells and human white blood cells. Cytokine release and their gene expressions of Jurkat cells and human white blood cells were measured by ELISA and reverse transcription polymerase chain reaction. TDI acutely promoted the interleukine-4 (IL-4) release significantly in both Jurkat cells and human white blood cells. TDI-induced IL-4 release was suppressed in the presence of 1,2-bis- (O-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), an intracellular Ca(2+) chelator, in Jurkat cells. In the hand of gene expression, TDI induced an increase in the mRNA level of TNF-alpha and IL-4 in Jurkat cells. We conclude that the release of IL-4 were coupled with the elevation in [Ca(2+)](c) induced by TDI. Further studies are required to clarify the roles of TDI-induced IL-4 secretion in acute inflammation.

Manganese regulation of virulence factors and oxidative stress resistance in Neisseria gonorrhoeae.

Neisseria gonorrhoeae has evolved a complex and novel network of oxidative stress responses, including defence mechanisms that are dependent on manganese (Mn). We performed systematic analyses at the transcriptomic and proteomic (1D SDS-PAGE and Isotope-Coded Affinity Tag [ICAT]) levels to investigate the global expression changes that take place in a high Mn environment, which results in a Mn-dependent oxidative stress resistance phenotype. These studies revealed that there were proteins regulated at the post-transcriptional level under conditions of increased Mn concentration, including proteins involved in virulence (e.g., pilin, a key adhesin), oxidative stress defence (e.g., superoxide dismutase), cellular metabolism, protein synthesis, RNA processing and cell division. Mn regulation of inorganic pyrophosphatase (Ppa) indicated the potential involvement of phosphate metabolism in the Mn-dependent oxidative stress defence. A detailed analysis of the role of Ppa and polyphosphate kinase (Ppk) in the gonococcal oxidative stress response revealed that ppk and ppa mutant strains showed increased resistance to oxidative stress. Investigation of these mutants grown with high Mn suggests that phosphate and pyrophosphate are involved in Mn-dependent oxidative stress resistance.

Biological inhibitory effects of the Chinese herb danggui on brain astrocytoma.

OBJECTIVE: Previous studies have demonstrated the utility of the traditional Chinese herb danggui in the treatment of chronic myelogenous leukemia. Our aim was to examine whether it might similarly be used to treat glioblastoma multiforme. METHODS: The lipid-soluble active ingredients of danggui were extracted with acetone (AS-AC) or chlorophenol (AS-CH) and their antiproliferative and proapoptotic effects were studiedin vitro on cultured GBM 8401 cells and in vivoon tumors in nude mice. RESULTS: After a 24-hour treatment, either AS-AC or AS-CH at a lower (50 micro g/ml) and a higher concentration (100 micro g/ml) significantly inhibited the proliferative activity of GBM 8401 cultured cells by 30-50%, as well as the expression of cathepsin B and vascular endothelial growth factor (VEGF). In nude mice, the growth of the tumor was inhibited by 30% by AS-CH or AS-AC (20 mg/kg; p < 0.05) and by 60% by AS-CH or AS-AC (60 mg/kg; p < 0.05). AS-AC and AS-CH also significantly inhibited microvessel formation in the tumors of nude mice. CONCLUSIONS: Danggui may inhibit tumor growth by reducing the level of VEGF and the proapoptotic protein, cathepsin B. Thus, danggui may be useful in the treatment of high-grade astrocytomas.

Increasing expression of extracellular matrix metalloprotease inducer in ovary tumors: tissue microarray analysis of immunostaining score with clinicopathological parameters.

Ovary cancer invasion is responsible for both local tissue destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell-derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesized that EMMPRIN isoverexpressed in ovary tumors. Immunohistochemical analysis of EMMPRIN was performed in tissue microarrays of ovary neoplasms including 84 cases of serous adenocarcinoma, 23 cases of mucinous adenocarcinoma, 10 cases of endometrioid adenocarcinoma, 12 cases of yolk sac tumor, 12 cases of clear cell carcinoma, 8 cases of dysgerminoma, 8 cases of granulosa cell tumor, 6 cases of transitional cell carcinoma, and 6 cases of Brenner tumor. All malignant ovary tumors showed significant immunohistochemical expression of EMMPRIN. The EMMPRIN scores in malignant ovary tumors were significantly higher than their nontumor counterparts (313+/-28 for serous adenocarcinoma; 308+/-25 for mucinous adenocarcinoma; 187+/-19 for endometrioid adenocarcinoma; 265+/-23 for yolk sac tumors; 87+/-13 for clear cellcarcinoma; 126+/-15 for dysgerminoma; 243+/-26 for granulosa cell tumor; 87+/-16 for transitional cell carcinoma). The EMMPRIN score was significantly higher in serous adenocarcinomas than in serous adenomas and serous borderline tumors and was correlated with nodal stage. Our findings show for the first time that EMMPRIN is overexpressed in all malignant ovary tumors.

Expression of serine protease matriptase in renal cell carcinoma: correlation of tissue microarray immunohistochemical expression analysis results with clinicopathological parameters.

Serine protease matriptase (matriptase) cleaves and activates proteins implicated in the progression of cancer and represents a potential therapeutic target. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 168 renal cell carcinomas (RCCs). All subtypes of RCC showed significant immunohistochemical expression of matriptase. In contrast, no expression occurred in areas of RCC with sarcomatous differentiation (SRCC) and in normal collecting tubules. The matriptase scores were significantly higher in papillary RCC (341+/-28) and clear cell RCC with granular cell differentiation (GRCC; 324+/-27) than in other histologic subtypes of RCC. In GRCC, matriptase scores were correlated with TNM staging and nuclear grading. Matriptase was overexpressed in all subtypes of RCC, and matriptase scores could distinguish between conventional clear cell RCC, GRCC, and SRCC.

Increasing expression of serine protease matriptase in ovarian tumors: tissue microarray analysis of immunostaining score with clinicopathological parameters.

Matriptase is a type II transmembrane serine protease expressed by cells of surface epithelial origin, including epithelial ovarian tumor cells. Matriptase cleaves and activates proteins implicated in the progression of cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine the expression of matriptase in ovarian tumors and to assign clinicopathological correlations. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 164 ovarian neoplasms including 84 serous adenocarcinomas, 23 mucinous adenocarcinomas, 10 endometrioid adenocarcinomas, six yolk sac tumors, 12 clear cell carcinomas, six dysgerminomas, eight granulosa cell tumors, four transitional cell carcinomas, five fibromas, and six Brenner tumors. All ovarian tumors except the fibromas and Brenner tumors showed significant expression of matriptase. The matriptase scores were significantly higher in the tumors than in their nontumor counterparts (304+/-26 for serous adenocarcinoma; 361+/-28 for mucinous adenocarcinoma; 254+/-17 for endometrioid adenocarcinoma; 205+/-19 for yolk sac tumor; 162+/-16 for clear cell carcinoma; 109+/-11 for dysgerminoma; 105+/-9 for granulosa cell tumor; and 226+/-18 for transitional cell carcinoma). Matriptase scores in serous adenocarcinoma were correlated with TNM stage and FIGO stage. Our findings demonstrate for the first time that matriptase is overexpressed in many malignant ovarian tumors. It may be a novel biomarker for diagnosis and treatment of malignant ovarian tumors.

Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses.

Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.

Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.

We have investigated the plasma proteome by using 2D gel electrophoresis and MS from patients with severe acute respiratory syndrome (SARS). A complete proteomic analysis was performed on four patients with SARS in different time courses, and a total of 38 differential spots were selected for protein identification. Most of the proteins identified are acute phase proteins, and their presence represents the consequence of serial cascades initiated by SARS-coronavirus infection. There are several proteins that have never been identified in plasma before using 2D gel electrophoresis, among which peroxiredoxin II was chosen for further study by analyzing additional 20 plasma samples from patients with probable and suspected SARS and patients with fever, respectively. The results showed that the level of plasma peroxiredoxin II in patients with SARS is significantly high and could be secreted by T cells. Taken together, our findings indicate that active innate immune responses, along with the oxidation-associated injuries, may play a major role in the pathogenesis of SARS.

IgA immune complex blunts the contraction of cultured mesangial cells through the inhibition of protein kinase C and intracellular calcium.

The effects of IgA immune complex (IgA-IC) on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with IgA-IC for 24 hours significantly decreased the contractile responses to angiotensin II (10(-6) M) and phorbol 12-myristate 13-acetate (PMA, 10(-6) M). Pretreatment of mesangial cells with the protein kinase C (PKC) inhibitor, chelerythrine (10(-6) M), eliminated the difference in contractile responses to angiotensin II or PMA between the control and IgA-IC groups indicating IgA-IC may inhibit the activity of PKC. The contractile responses to ionomycin were not significantly different between IgA-IC treated and control mesangial cells, suggesting that the contractile machinery is not impaired by IgA-IC. Intracellular calcium, [Ca2+]i measured by changes in fura-2 level in response to ATP or bradykinin, was significantly inhibited in IgA-IC treated mesangial cells, compared to control cells. In contrast, treatment with thapsigargin did not result in significant differences in [Ca2+]i between IgA-IC and control mesangial cells, suggesting that a negligible role of endoplasmic reticulum in the effects of IgA-IC. Using PKC specific antibodies, IgA-IC significantly increased the particulate fraction of PKC-iota of mesangial cells to 141+/-13% of control, without significantly changing the protein content of PKC-alpha, -delta and -lambda in the cytosolic and particulate fractions. In summary, IgA-IC inhibits the contractile responses of cultured mesangial cells to agonists by inhibiting the activation of PKC and [Ca2+]i.

Adriamycin impairs the contraction of mesangial cells through the inhibition of protein kinase C and intracellular calcium.

The effects of adriamycin on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with adriamycin (0.2 microg/ml) for 24 h significantly decreased the contractile responses to the calcium channel activator BAY K 8644 (1 microM) and to the PKC activator PMA (1 microM). Intracellular calcium concentration ([Ca(2+)](i)), measured by changes in fura 2 levels in response to ATP (0.1 mM), was significantly inhibited in adriamycin-treated mesangial cells compared with control cells. In the absence of extracellular calcium, treatment with ionomycin (0.1 mM) or thapsigargin (10 microM) resulted in a significantly smaller increase in [Ca(2+)](i) in adriamycin-treated mesangial cells compared with control, suggesting an important role of the endoplasmic reticulum in the effects of adriamycin. Using PKC-specific antibodies, adriamycin significantly decreased the cytosolic and membranous fractions of PKC-alpha in mesangial cells to 75 +/- 6 and 70 +/- 12% of control, respectively. The PKC activity of mesangial cells was also significantly inhibited after incubation with adriamycin for 24 h. In conclusion, adriamycin induces hypocontractility of mesangial cells, which may mediate this effect by inhibiting PKC-alpha and [Ca(2+)](i).

A model to study neurotropism and persistency of Japanese encephalitis virus infection in human neuroblastoma cells and leukocytes.

Japanese encephalitis (JE) is the most common mosquito-borne encephalitis in the Asia-Pacific region. Patients with JE usually present neuronal involvement, but other organ involvement is relatively rare. Employing human neuroblast-derived (NB) cell lines and different blood cells (erythrocytes, lymphocytes, granulocytes and monocytes), the neurotropism and persistency of Japanese encephalitis virus (JEV) in human cells was investigated. It was found that JEV could not replicate in erythrocytes, granulocytes or lymphocytes. Monocytes and NB cell lines could support replication of JEV as demonstrated by expression of viral NS3 antigen and virus plaque-forming units (p.f.u.). JEV could replicate more efficiently in neuroblastoma (HTB-11) cells than in monocytes after infection for 48 h (2.1+/-1.2x10(7) vs 2.8+/-0.7x10(2) p.f.u. ml(-1)). Two different strains of JEV revealed a similar infectivity to different leukocytes and four NB cell lines. In a kinetic study, it was found that JEV-infected monocytes possessed a high viability (90 %) after infection for 5 days, while JEV-infected neuroblastoma cells suffered cell apoptosis in 2 days and decreased viability to less than 1 % in 5 days. Further studies showed that monocytes could take up JEV rapidly, displaying a log scale increase of intracellular JEV titres in 9 h after infection. Significantly, extracellular production of JEV by monocytes started in 12 h, peaked in 3 days and persisted for more than 3 weeks. These results suggest that JEV-infected monocytes may play an important role in harbouring JEV for eventual transmission to NB cells and that modulation of JEV-induced NB cell apoptosis may be useful in treating patients with JE.

Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus.

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6-9, and mutation of the second one (KKSK), located at aa 73-76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85-100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

BACKGROUND: Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. RESULTS: The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation). CONCLUSIONS: Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.