Sho Tone Lee

Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages

Although capsaicin analogs might be a potential strategy to manipulate inflammation, the mechanism is still unclear. In this study, the effects and action mechanisms of vanilloid analogs on iNOS and COX‐2 expression were investigated in RAW264.7 macrophages. Capsaicin and resiniferatoxin (RTX) can inhibit LPS‐ and IFN‐γ‐mediated NO production, and iNOS protein and mRNA expression with similar IC50 values of around 10 μM. Capsaicin also transcriptionally inhibited LPS‐ and PMA‐induced COX‐2 expression and PGE2 production. However, this effect exhibited a higher potency (IC50: 0.2 μM), and RTX failed to elicit such responses at 10 μM. Interestingly, we found that capsazepine, a competitive TRPV1 antagonist, did not prevent the inhibition elicited by capsaicin or RTX. Nevertheless, it mimicked vanilloids in inhibiting iNOS/NO and COX‐2/PGE2 induction with an IC50 value of 3 μM. RT–PCR and immunoblotting analysis excluded the expression of TRPV1 in RAW264.7 macrophages. The DNA binding assay demonstrated the abilities of vanilloids to inhibit LPS‐elicited NF‐κB and AP‐1 activation and IFN‐γ‐elicited STAT1 activation. The reporter assay of AP‐1 activity also supported this action. The kinase assay indicated that ERK, JNK, and IKK activation by LPS were inhibited by vanilloids. In conclusion, vanilloids can modulate the expression of inflammatory iNOS and COX‐2 genes in macrophages through interference with upstream signalling events of LPS and IFN‐γ. These findings provide new insights into the potential benefits of the active ingredient in hot chilli peppers in inflammatory conditions.

Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum.

The polysaccharide from Ganoderma lucidum (PS‐G) has been reported to enhance immune responses and to elicit antitumor effects. In our previous study, we found that PS‐G efficiently inhibited spontaneously and Fas‐enhanced neutrophil apoptosis when cultured in vitro. Since phagocytosis and chemotaxis play essential roles in host defense mediated by neutrophils, it is of great interest to know the effect of PS‐G on these two cell functions, and the molecular events leading to these actions. Using latex beads and heat‐inactive Escherichia coli serving as particles for neutrophil engulfment, we found that PS‐G is able to enhance phagocytic activity of human primary neutrophils and neutrophilic‐phenotype cells differentiated from all trans retinoic acid‐treated HL‐60 cells. Chemotactic assay using Boyden chamber also revealed the ability of PS‐G to increase neutrophil migration. Exposure of neutrophils to PS‐G time dependently caused increases in protein kinase C (PKC), p38 mitogen‐activated protein kinase (MAPK), Hck, and Lyn activities. Results with specific kinase inhibitors indicate that phagocytic action of PS‐G was reduced by the presence of wortmannin (Phosphatidylinositol 3‐kinase, PI3K inhibitor), pyrazolpyrimidine 2 (Src‐family tyrosine kinase inhibitor), Ro318220 (PKC inhibitor), and SB203580 (p38 MAPK inhibitor), but not by PD98059 (mitogen‐activated protein/ERK kinase inhibitor). Moreover, chemotactic action of PS‐G requires the activities of PI3K, p38 MAPK, Src tyrosine kinases and PKC. All these results demonstrate the abilities of PS‐G to enhance neutrophil function in phagocytosis and chemotaxis, and further provide evidence to strengthen the beneficial remedy of G. lucidum in human to enhance defense system.

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

Characterization of sequence changes in kinetoplast DNA maxicircles of drug-resistant Leishmania

We have compared kinetoplast DNA maxicircles of tunicamycin- and arsenite-resistant variants of repeatedly cloned Leishmania mexicana amazonensis showing DNA amplification with wild-type and arsenite-resistant variants of the same lineage that do not show DNA amplification. DNA restriction patterns and the degree of cross-hybridization between maxicircle DNA fragments of parasites displaying DNA amplification and those of parasites without amplification were examined. In addition, the nucleotide sequence of the cytochrome b (Cyb) gene from the coding region was compared between these two groups of parasites. Extensive changes were found in the nucleotide sequences and the amino acid sequences of the cytochrome gene of the maxicircles of variants with DNA amplification. The Cyb genes from both groups had much shorter open reading frames than the same gene from Leishmania tarentolae and Trypanosoma brucei. The simultaneous changes in maxicircles and minicircles of these variants suggest that they may confer the advantage of maintaining viable mitochondrial function under selective pressure.

Bcl3 Bridges LIF‐STAT3 to Oct4 Signaling in the Maintenance of Naïve Pluripotency

Leukemia inhibitory factor (LIF) regulates mouse embryonic stem cell (mESC) pluripotency through STAT3 activation, but the downstream signaling remains largely unelucidated. Using cDNA microarrays, we verified B cell leukemia/lymphoma 3 (Bcl3) as the most significantly downregulated factor following LIF withdrawal in mESCs. Bcl3 knockdown altered mESC morphology, reduced expression of pluripotency genes including Oct4, Sox2, and Nanog, and downregulated DNA binding of acetylated histone 3 and RNA polymerase II on the Oct4 promoter. Conversely, Bcl3 overexpression partially prevented cell differentiation and promoted Oct4 and Nanog promoter activities. Furthermore, coimmunoprecipitation and chromatin immunoprecipitation experiments demonstrated that Bcl3 regulation of mESC pluripotency may be through its association with Oct4 and β‐catenin and its promoter binding capability. These results establish that Bcl3 positively regulates pluripotency genes and thus shed light on the mechanism of Bcl3 as a downstream molecule of LIF/STAT3 signaling in pluripotency maintenance. Stem Cells 2015;33:3468–3480

Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

Indigenous leishmaniasis in Taiwan: report of a case.

Taiwan is not considered an endemic area of leishmaniasis. Imported cases are encountered infrequently, and only two cases of indigenous cutaneous leishmaniasis have been reported. 1 We found one new case in the past 20 years. The patient presented with erythematous plaques on the nasal bridge and right thumb. Skin biopsy specimens from both sites revealed numerous Leishman–Donovan bodies in macrophages. There was no history of travel outside the country, and the diagnosis of indigenous cutaneous leishmaniasis was made. Polymerase chain reactions (PCR) identified the species as Leishmania tropica. The route of infection in this patient is unclear. Because pentavalent antimony, the drug of choice for leishmaniasis, is not available in Taiwan, the patient was treated with levamisole and potassium iodide, with an excellent response.

Identification of macrosialin (CD68) on the surface of host macrophages as the receptor for the intercellular adhesive molecule (ICAM-L) of Leishmania amazonensis

The intercellular adhesive molecule, ICAM-L, of Leishmania amazonensis is known to block the attachment as well as internalisation of Leishmania for infection in host macrophages. We employed monoclonal antibodies (mAb) to the surface molecules of a macrophage to block the attachment of ICAM-L to the macrophage surface and identified that CD68 macrosialin is likely the receptor molecule on the macrophage for ICAM-L. We then demonstrated physical interaction between ICAM-L and macrosialin by co-immunoprecipitation of macrosialin with ICAM-L or vice versa. Finally, macrosialin is expressed in macrosialin-negative murine fibroblast cell line NCTC clone 2555 and demonstrates that both ICAM-L and promastigotes of L. amazonensis can bind to the CD68 transfectant. We thus conclude that CD68 macrosialin is the receptor on host macrophages for ICAM-L. Also, involvement of ICAM-L-macrosialin interaction in other Leishmania species and other mammalian macrophages were demonstrated, indicating the biological relevance of this ligand-receptor interaction.

An improved method for detection of Leishmania amastigotes by an antibody probe against the small subunit of leishmanial ribonucleotide reductase

Abstract By taking advantage of an antibody raised against the small M2 subunit of ribonucleotide reductase of Leishmania that reacts with the enzyme in the nucleus of the parasite but does not cross-react with the same enzyme of the host macrophage, an improved fluorescence-staining method is developed for enumeration of leishmanial amastigotes inside the macrophage. The method offers an accurate and easy way of counting, compared with Giemsa staining.

MYASTHENIA GRAVIS ACCOMPANIED BY THYMOMAS NOT RELATED TO FOAMY VIRUS GENOME IN BELARUSIAN'S PATIENTS

The spectrum and features of neurological disorders have been changed due to the Chernobyl catastrophe in the Republic of Belarus. More recently neurologists in Belarus have noted a significant increase in the frequency of myasthenia gravis (MG) with concomitant rise in the thymomas. There is some evidence suggesting that retroviruses play a key role in the development and pathogenesis of autoimmune diseases. This study analyzed thymomas from 45 MG patients from the Republic of Belarus by using PCR and primers for two regions of FV—gag and bel-2 genes. The results showed that none of the varied thymuses from the 45 MG patients contained FV genome. No relationship can be confirmed between FV and this disease and the results suggest that no pathological link between FV and MG exists.