In vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, and Phellodendron cortex.

BACKGROUND: A search for new anti-coronaviral drugs to treat coronaviral infections was motivated by an outbreak of severe acute respiratory syndrome (SARS). OBJECTIVES: In order to find drugs that treat coronavirus infections, including SARS, we screened traditional medicinal herbal extracts and evaluated their antiviral activities on coronavirus replication. STUDY DESIGN: We employed a plaque assay to evaluate the effect of 22 medicinal herbal extracts on virus replication. We determined the 50% effective concentration (EC50) of each extract that was necessary to inhibit the replication of mouse hepatitis virus A59 (MHV-A59); we also determined 50% cytotoxic concentrations (CC50) for each extract. Northern and Western blot analyzes were performed to investigate antiviral activity in MHV-infected DBT cells, including virus entry, viral RNA and protein expression, and virus release. Coronavirus specific inhibition was also demonstrated using porcine epidemic diarrhea virus (PEDV). RESULTS: Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, Phellodendron cortex and Sophora subprostrata radix decreased the MHV production and the intracellular viral RNA and protein expression with EC50 values ranging from 2.0 to 27.5 microg/ml. These extracts also significantly decreased PEDV production and less dramatically decreased vesicular stomatitis virus (VSV) production in vitro. CONCLUSIONS: The extracts selected strongly inhibited MHV replication and could be potential candidates for new anti-coronavirus drugs.     

Food-anticipatory activity and liver per1-luc activity in diabetic transgenic rats

The mammalian Per1 gene is an important component of the core cellular clock mechanism responsible for circadian rhythms. The rodent liver and other tissues rhythmically express Per1 in vitro but typically damp out within a few cycles. In the liver, the peak of this rhythm occurs in the late subjective night in an ad lib-fed rat, but will show a large phase advance in response to restricted availability of food during the day. The relationship between this shift in the liver clock and food-anticipatory activity (FAA), the circadian behavior entrained by daily feeding, is currently unknown. Insulin is released during feeding in mammals and could serve as an entraining signal to the liver. To test the role of insulin in the shift in liver Per1 expression and the generation of FAA, per-luciferase transgenic rats were made diabetic with a single injection of streptozotocine. Following 1 week of restricted feeding and locomotor activity monitoring, liver was collected for per-luc recording. In two separate experiments, FAA emerged and liver Per1 phase-shifted in response to daytime 8-h food restriction. The results rule out insulin as a necessary component of this system.     

Two cases of lichen planus pigmentosus presenting with a linear pattern

We report two cases of lichen planus pigmentosus (LPP) that developed in a unilateral linear pattern. The patients presented with unilateral linear brown macules on the extremities. Skin biopsy showed orthokeratosis, basal hydropic degeneration with scarce lymphohistiocytic infiltrates, and numerous melanophages in both patients. These patients, to the best of our knowledge, are the first cases of LPP presenting with a linear pattern. LPP should be considered in the differential diagnosis of linear hyperpigmented skin lesions.     

Isolation of endothelial progenitor cells from cord blood and induction of differentiation by ex vivo expansion

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1 beta , fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.     

Protective effect of Hypericum perforatum Linn (St. John's wort) against hydrogen peroxide-induced apoptosis on human neuroblastoma cells

The medicinal plant Hypericum perforatum Linn, commonly known as St. John's wort, has been used as an antidepressant. To investigate whether St. John's wort possesses a protective effect against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in neuronal cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 4,6-diamidino-2-phenylindole staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, flow cytometry analysis, DNA fragmentation assay, and caspase-3 enzyme assay were performed on SK-N-MC human neuroblastoma cells. Cells treated with H(2)O(2) exhibited several apoptotic features, while those pre-treated with St. John's wort prior to H(2)O(2) exposure showed a decreased occurrence of apoptotic features. In addition, pre-treatment with St. John's wort inhibited H(2)O(2)-induced increase in caspase-3 enzyme activity. These results suggest that St. John's wort may exert a protective effect against H(2)O(2)-induced apoptosis in human neuroblastoma cells.     

No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

Activation of extracellular signal-regulated kinases in the sciatic nerves of rats with experimental autoimmune neuritis

To investigate whether the phosphorylation of extracellular signal-regulated kinase (ERK) is involved in autoimmune injury of the peripheral nervous system (PNS), the expression of phosphorylated ERK (p-ERK) was analyzed in experimental autoimmune neuritis (EAN) in rats. Western blot analysis showed that the level of p-ERK was increased significantly in the sciatic nerves of rats on days 14 (p<0.05) and 24 (p<0.01) post-immunization, compared with controls, and its reaction declined at day 30 post-immunization. Immunohistochemistry showed that p-ERK protein was weakly expressed in Schwann cells and vascular endothelial cells in the sciatic nerves of CFA-immunized control rats. In EAN-affected sciatic nerves, p-ERK immunoreactivity was found mainly in ED1-positive macrophages on days 14 and 24 post-immunization. Moreover, on days 24 and 30 post-immunization, p-ERK immunoreactivity increased gradually in the Schwann cells of rat sciatic nerves with EAN. Based on these results, we postulated that the phosphorylation of ERK has an important role in the differentiation and survival of cells, including inflammatory cells and Schwann cells, in the rat sciatic nerve in EAN. Specifically, the activation of ERK in the recovery phase of EAN paralysis seems to be related in the survival of Schwann cells.     

Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10(8) CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60 degrees C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.     

Role of hydrophobic interaction in the enantioselective solvolyses of amino acid p-nitrophenyl esters by optically active imidazole-containing polymers

Solvolytic reactions of N-benzyloxycarbonyl-D -phenylalanine p-nitrophenyl ester and N-benzyloxycarbonyl-L -phenylalanine p-nitrophenyl ester by optically active imidazole-containing polymers were performed at different temperatures and in aq. ethanol of different water contents. The catalyst polymers employed were homopolymers of N-methacryloyl-L -histidine ( 1a ) and N-methacryloyl-L -histidine methyl ester ( 1b ), as well as copolymers of 1a with dodecyl methacrylate (DMA) and 1b with DMA. In 30 vol. -% aqueous ethanol at pH 7,02 the homopolymers did not show any enantioselective catalysis. However, the copolymers did exhibit enantioselective catalysis, viz., k_cat(L )/k_cat(D ) = 1,25 for poly ( 1b -co-DMA) containing 5,7 mol-% of DMA. As the reaction temperature was lowered, the reaction rate increased and the enantioselectivity was enhanced (k_cat(L )/k_cat(D ) = 1,67 for poly ( 1b -co-DMA) at 10°C). When the ethanol content was decreased, enhanced reaction rates and enantioselectivity (k_cat(L )/k_cat(D ) = 1,65 for poly ( 1 b -co-DMA) in 20 vol.-% aqueous ethanol) were observed. From these results it is concluded that hydrophobic interaction plays an important role in the enantioselective catalysis of optically active imidazole-containing polymers.