The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Synthesis, in vitro assay, and molecular modeling of new piperidine derivatives having dual inhibitory potency against acetylcholinesterase and Abeta1-42 aggregation for Alzheimer's disease therapeutics

With the goal of developing Alzheimer's disease therapeutics, we have designed and synthesized new piperidine derivatives having dual action of acetylcholinesterase (AChE) and beta-amyloid peptide (Abeta) aggregation inhibition. For binding with the catalytic site of AChE, an ester with aromatic group was designed, and for the peripheral site, another aromatic group was considered. And for intercalating amyloid-beta oligomerization, long and linear conformation with a lipophilic group was considered. The synthetic methods employed for the structure with dual action depended on alcohols with an aromatic ring and the substituted benzoic acids, which are esterificated in the last step of the synthetic pathway. We screened these new derivatives through inhibition tests of acetylcholinesterase, butyrylcholinesterase (BChE), and Abeta(1-42) peptide aggregation, AChE-induced Abeta(1-42) aggregation. Our results displayed that compound 12 showed the best inhibitory potency and selectivity of AChE, and 29 showed the highest selectivity of BChE inhibition. Compounds 15 and 12 had inhibitory activities against Abeta(1-42) aggregation and AChE-induced Abeta aggregation. In the docking model, we confirmed that 4-chlorobenzene of 12 plays the parallel pi-pi stacking against the indole ring of Trp84 in the bottom gorge of AChE. Because the benzyhydryl moiety of 12 covered the peripheral site of AChE in a funnel-like shape, 12 showed good inhibitory potency against AChE and could inhibit AChE-induced Abeta(1-42) peptide aggregation.     

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.     

Analysis of promoter methylation and polymorphic minisatellites of BORIS and lack of association with gastric cancer

BORIS is a member of the cancer-testis gene family that comprises genes normally expressed only in testis but abnormally activated in different malignancies. In this study, we examined the relation between BORIS expression and gastric cancer, which is the most common cancer in Korea. Abnormal BORIS expression in the patient's gastric cancer tissues was observed. We checked the methylation status of the gene in gastric cancer tissue, because the regulation by methylation in its CpG islands is well known for BORIS. However, there was no correlation between the methylation status and gene expression. Then, we focused on the minisatellites (variable number of tandem repeats) of BORIS as another possible regulator for this abnormal expression. Previously, we reported the characterization of BORIS-MS2 and determined the frequency of alleles in cancer patients. A case-control study was performed using DNA from 774 controls and 496 patients with gastric cancer. There was no significant difference observed in the overall distribution of minisatellite alleles. These results suggest that additional different regulators for the abnormal BORIS expression in gastric cancer may exist. Additionally, we performed a segregation analysis of BORIS-MS2 with genomic DNA obtained from two generations of five families and from three generations of two families. BORIS-MS2 alleles were transmitted through meiosis following Mendelian inheritance, which suggests that this polymorphic minisatellite could be a useful marker for paternity mapping and DNA fingerprinting.     

Variants of BORIS minisatellites and relation to prognosis of prostate cancer

BORIS, a member of the cancer-testis antigen family with testis specific expression, showed abnormal expression in various cancer types including prostate cancer. In our previous work, we identified the polymorphic minisatellite, BORIS-MS2, located in the promoter region of BORIS. BORIS-MS2 was revealed as a polymorphic minisatellite that contains seven alleles each with different numbers of the repeat unit. We assessed the association between the allelic variation of BORIS-MS2 and prostate cancer by a case-control study. Using a PCR-based method, 315 cancer-free male controls and 648 cases with prostate cancer were genotyped. There was no significant difference observed in the overall distribution of this minisatellite, which indicates that this polymorphism is not responsible for prostate cancer susceptibility in the Korean population. Additionally, two new rare alleles (9 and 19 copies) were detected in this study, which were identified only in cancer subjects. When we compared the clinicopathological information with the Jewett-Whitmore system of prostate cancer classification, the frequency of rare allele cases in the higher grade was significantly higher than in the total prostate group (P = 0.032). The higher grades in the Jewett-Whitmore system were associated with a poor prognosis. Therefore, this result suggests that the rare alleles of BORIS-MS2 may be used as a marker of poor outcome in prostate cancer patients.     

A novel hypothesis for the binding mode of HERG channel blockers

We present a new docking model for HERG channel blockade. Our new model suggests three key interactions such that (1) a protonated nitrogen of the channel blocker forms a hydrogen bond with the carbonyl oxygen of HERG residue T623; (2) an aromatic moiety of the channel blocker makes a pi-pi interaction with the aromatic ring of HERG residue Y652; and (3) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. The previous model assumes two interactions such that (1) a protonated nitrogen of the channel blocker forms a cation-pi interaction with the aromatic ring of HERG residue Y652; and (2) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. To test these models, we classified 69 known HERG channel blockers into eight binding types based on their plausible binding modes, and further categorized them into two groups based on the number of interactions our model would predict with the HERG channel (two or three). We then compared the pIC(50) value distributions between these two groups. If the old hypothesis is correct, the distributions should not differ between the two groups (i.e., both groups show only two binding interactions). If our novel hypothesis is correct, the distributions should differ between Groups 1 and 2. Consistent with our hypothesis, the two groups differed with regard to pIC(50), and the group having more predicted interactions with the HERG channel had a higher mean pIC(50) value. Although additional work will be required to further validate our hypothesis, this improved understanding of the HERG channel blocker binding mode may help promote the development of in silico predictions methods for identifying potential HERG channel blockers.     

Characterization of thermostable deblocking aminopeptidases of archaeon Thermococcus onnurineus NA1 by proteomic and biochemical approaches

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.     

Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes

Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPC(PSA-NCAM+)) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPC(PSA-NCAM+) extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80～98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPC(PSA-NCAM+) showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPC(PSA-NCAM+) differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPC(PSA-NCAM+) compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.     

Correlation of IGF1R expression with ABCG2 and CD44 expressions in human osteosarcoma

Osteosarcoma is the most common type of malignant bone tumors. Insulin Growth Factor 1 receptor (IGFR1) has been known as a prognostic factor for metastasis of osteosarcoma. ABC subfamily G member2 (ABCG2) is related to resistance to anti-cancer drug, and CD44 has a role in tumor growth and metastasis. The purpose of this study is to investigate the relationship among expression patterns of IGF1R, ABCG2, and CD44 in osteosarcoma. The expression levels of IGF1R, ABCG2, and CD44 proteins were determined in tissue arrays containing osteosarcoma tissues from 59 osteosarcoma patients. The expression pattern of IGF1R was highly correlated with the expression pattern of ABCG2 (r?=?0.88) in overall osteosarcoma patients. According to pathological types, the expression pattern of IGF1R showed the higher correlation with ABGC2 (r?=?0.90) and CD44 (r?=?0.61) in osteoblatic type than in chondroblastic type. According to gender with pathologic type, the correlation between the expression patterns of IGF1R and CD44 was higher in male with osteoblatic type than in female with osteoblatic type. Among different age groups, the 1–10 years age group showed higher correlation in IGF1R versus CD44 (r?=?0.90) and ABCG2 versus CD44 (0.80) than in other age groups. These results showed that the expression of IGF1R appears to be highly correlated with the expression of ABCG2 in osteosarcoma and with the expression of CD44 in osteosarcoma patients under age of 10, which suggests that ABCG2 and CD44 can be used as prognostic factors with IGF1R for specific prognosis and efficient treatment of osteosarcoma.