Ming Chiu Fung

Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response

Dying primary liver, NIH 3T3, and HeLa cells can reverse the advanced stage of apoptosis and survive even after incurring DNA damage. Some surviving cells harbor genetic alterations that result in phenotypic diversity, including oncogenic transformation.

Vimentin supports mitochondrial morphology and organization

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.

Cucurbitacin B induces differentiation, cell cycle arrest, and actin cytoskeletal alterations in myeloid leukemia cells

Cucurbitacins have long been utilized for their abortifacient and anti-inflammatory effects; however, little is known about their mechanism of action. In this study, we have demonstrated robust antiproliferative effects of CuB on various leukemia and lymphoma cell lines, as well as on primary mononuclear bone marrow cells derived from patients with acute myeloid leukemia or myelodysplastic syndrome. Myeloid leukemic cells treated with CuB exhibit significant S-phase cell cycle arrest, enlarged cell size, multinucleation, and enhanced expression of a monocytic- and granulocytic-specific CD11b. Moreover, our data demonstrate that CuB prominently alters the cytoskeletal network of leukemic cells, inducing rapid and improper polymerization of the F-actin network. These encouraging results suggest the appropriateness of clinical trials of cucurbitacins for the treatment of hematopoietic malignancies.

Reversibility of apoptosis in cancer cells

Apoptosis is a cell suicide programme characterised by unique cellular events such as mitochondrial fragmentation and dysfunction, nuclear condensation, cytoplasmic shrinkage and activation of apoptotic protease caspases, and these serve as the noticeable apoptotic markers for the commitment of cell demise. Here, we show that, however, the characterised apoptotic dying cancer cells can regain their normal morphology and proliferate after removal of apoptotic inducers. In addition, we demonstrate that reversibility of apoptosis occurs in various cancer cell lines, and in different apoptotic stimuli. Our findings show that cancer cells can survive after initiation of apoptosis, thereby revealing an unexpected potential escape mechanism of cancer cells from chemotherapy.

Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. b. brucei infection in mice

The beta‐tubulin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi beta‐tubulin shows 100%, 99·8%, 99·1%, and 98·6% homology with T. equiperdum, T. b. brucei, T. cruzi and T. danilewskyi, respectively, but is diverse from that of T. cyclops, showing only 51·6% of homology. Recombinant beta‐tubulin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant beta‐tubulin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818 and T. b. brucei STIB 940, showing 83·3%, 70% and 76·7% protection, respectively. Serum collected from the rabbit immunized with recombinant beta‐tubulin inhibited the growth of T. evansi, T. equiperdum and T. b. brucei in vitro. Serum from mice and rabbits immunized with recombinant beta‐tubulin recognized only T. evansi beta‐tubulin and not mouse beta‐tubulin. The results of this study demonstrated that the recombinant T. evansi beta‐tubulin is a potential candidate for the development of a vaccine to prevent animal trypanosomiasis caused by these three trypanosome species.

In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity

The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

Immune mechanisms associated with the rejection of fetal murine proislet allografts and pig proislet xenografts: comparison of intragraft cytokine mrna profiles1

BACKGROUND Previous in vivo depletion studies of CD4 and CD8 T cells indicated that different rejection mechanisms operate for proislet allografts and xenografts. The cellular and molecular mechanisms of acute proislet allograft and xenograft rejection have therefore been characterized and directly compared. METHODS The intragraft cytokine mRNA profile in rejecting BALB/c (H-2d) proislet allografts was analyzed in control, CD4 T cell-depleted, and CD8 T cell-depleted CBA/H (H-2k) recipient mice using semi-quantitative reverse transcriptase-assisted polymerase chain reaction (RT-PCR). The cytokine profiles for proislet allografts and pig proislet xenografts at 3-10 days posttransplant were directly compared and correlated with graft histopathology. RESULTS Allograft rejection was protracted (2-3 weeks), characterized by infiltrating CD8 T cells and CD4 T cells (no eosinophils) and was associated with a Th1-type CD4 T cell response (IL-2, IFN-gamma, and IL-3 mRNA) and a CD8 T cell-dependent spectrum of cytokine gene expression (IL-2, IFN-gamma, IL-3, and IL-10 mRNA). Xenograft rejection was rapid (6-8 days), involved predominantly CD4 T cells and eosinophils, and in contrast to allografts, exhibited intragraft mRNA expression for the Th2 cytokines IL-4 and IL-5. CONCLUSIONS Proislet allograft and xenograft rejection differ in the tempo of destruction, phenotype of the cellular response and intragraft profile of cytokine mRNA. The recruitment of eosinophils only to the site of xenorejection correlates with IL4 and IL-5 mRNA expression. These findings suggest that different anti-rejection strategies may need to be developed to optimally target the allograft and the xenograft response.

Schistosoma japonicum : proteomics analysis of differentially expressed proteins from ultraviolet-attenuated cercariae compared to normal cercariae

Schistosomiasis is considered the most important human helminthiasis in terms of morbidity and mortality. In this study, comparative soluble proteomic analysis of normal cercariae and ultraviolet-irradiated attenuated cercariae (UVAC) from Schistosoma japonicum were carried out in view of the high efficiency of irradiation-attenuated cercariae vaccine. The results revealed that some proteins showed significant differential expression in the parasite after treatment with ultraviolet light. Total 20 protein spots were identified by mass spectrometry, corresponded to five groups according to their functions in the main that were structural and motor proteins (actin, et al.), energy metabolism associated enzymes (glyceraldehydes-3-phosphage dehydrogenase, et al.), signaling transduction pathway-associated molecules (14-3-3 protein, et al.), heat shock protein families (HSP 70 family, et al.), and other functional proteins (20S proteasome). Furthermore, our results indicated that the differential expression of the proteins by ultraviolet irradiation may be, at least partially, acquired by regulating the mRNA levels of corresponding proteins. These results may provide new clues for further exploring the mechanism of protective immunity induced by UVAC and may shed some light on the development of vaccines against schistosomiasis.

Molecular cloning and expression of a functional anti-inflammatory protein, Sj16, of Schistosoma japonicum.

Schistosomes are the causative agent of schistosomiasis. In the infected host, significant inflammatory response to the parasite is not observed. Previous studies of Schistosoma mansoni showed that this subdued inflammatory response was due to a 16-kDa protein, Sm16, which is present in high levels in the secretions of schistosomula. Here we report the cloning and characterization of a gene (named Sj16) from Schistosoma japonicum. Sequence analysis showed that Sj16 shares 99% identity with Sm16 in its nucleotide sequence, and 100% identity in its protein sequence. While previous studies reportedly failed to obtain the soluble recombinant protein of Sm16, we expressed and purified recombinant Sj16 (rSj16) from Escherichia coli. Western blot and ELISA analyses showed that S. japonicum-infected rabbit sera could not recognize rSj16, indicating that native Sj16 may fail to induce circulating antibodies during S. japonicum infection. In vivo, rSj16 dramatically suppressed the recruitment of thioglycollate-mediated leukocytes to the peritoneal cavity of BALB/c mice, accompanied by marked up-regulation of IL-10 and IL-1RA transcripts, and down-regulation of IL-12p35, IL-1 beta and MIP-2 transcripts in peritoneal cells. Further analysis revealed that rSj16 also suppressed thioglycollate-induced peritoneal macrophage maturation. These results demonstrate that rSj16 has an anti-inflammatory function.

Induction of Shrimp Tropomyosin-Specific Hypersensitivity in Mice

Background: Shellfish hypersensitivity is amongst the most common food allergies. The major shellfish allergen was identified as tropomyosin. Here, we investigated the immediate hypersensitivity responses, IgE and cell-mediated immune response in mice sensitized with recombinant shrimp tropomyosin. Methods: Shrimp tropomyosin was cloned and expressed as a His-tagged fusion recombinant protein in Escherichia coli. Three- to 4-week-old BALB/c mice were sensitized by intragastric administration of recombinant tropomyosin (0.1 mg) plus cholera toxin (10 µg) on days 0, 12, 19 and 26 and challenged on day 33. Mice fed with phosphate-buffered saline plus cholera toxin were included as controls. Animals were monitored for immediate hypersensitive responses and tropomyosin-specific IgE over time. In addition, shrimp tropomyosin-specific CD4+ T cells, interleukin-4 and interferon-γ levels were determined from in vitro splenocyte cultures. A passive cutaneous anaphylaxis assay was also conducted. Results: Mice fed with shrimp tropomyosin developed swelling of the snout, increased scratching behavior and shrimp tropomyosin-specific IgE. Sera from tropomyosin-sensitized mice elicited vascular leakage in naïve mice in the passive cutaneous anaphylaxis assay. Shrimp tropomyosin-specific CD4+ T cell proliferations and elevated interleukin-4 over interferon-γ levels were evident in splenocyte cultures of tropomyosin-fed mice upon tropomyosin stimulation. In contrast, shrimp tropomyosin-specific IgE, CD4+ T cells and hypersensitive responses were absent in the control mice. Conclusion: We have generated a BALB/c model of shrimp allergy. This model provides a useful tool for evaluating the immunopathogenic mechanisms involved in shellfish hypersensitivity.