Exceptional appendage and soft-tissue preservation in a Middle Triassic horseshoe crab from SW China.

Horseshoe crabs are classic "living fossils", supposedly slowly evolving, conservative taxa, with a long fossil record back to the Ordovician. The evolution of their exoskeleton is well documented by fossils, but appendage and soft-tissue preservation is extremely rare. Here we analyse details of appendage and soft-tissue preservation in Yunnanolimulus luopingensis, a Middle Triassic (ca. 244 million years old) horseshoe crab from Yunnan Province, SW China. The remarkable preservation of anatomical details including the chelicerae, five pairs of walking appendages, opisthosomal appendages with book gills, muscles, and fine setae permits comparison with extant horseshoe crabs. The close anatomical similarity between the Middle Triassic horseshoe crabs and their recent analogues documents anatomical conservatism for over 240 million years, suggesting persistence of lifestyle. The occurrence of Carcinoscorpius-type claspers on the first and second walking legs in male individuals of Y. luopingensis indicates that simple chelate claspers in males are plesiomorphic for horseshoe crabs, and the bulbous claspers in Tachypleus and Limulus are derived.

Presenilins regulate the cellular level of the tumor suppressor PTEN.

Alzheimer's Disease (AD) is characterized by amyloid plaques consisting of beta-amyloid (Abeta) peptides and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Abeta is proteolytically derived from its precursor protein through cleavages by beta-secretase and gamma-secretase complex comprising presenilins (PS, PS1/PS2), nicastrin, APH-1 and PEN-2. PS1 is also known to activate the PI3K/Akt cell survival pathway in a gamma-secretase-independent manner. The tumor suppressor PTEN, which antagonizes the PI3K/Akt pathway, has increasingly been recognized to play a key role in neural functions and its level found reduced in AD brains. Here, we demonstrate that the protein level of PTEN is dramatically reduced in cultured cells and embryonic tissues deficient in PS, and in the cortical neurons of PS1/PS2 conditional double knockout mice. Restoration of PS in PS-deficient cells reverses the reduction of PTEN. Regulation of PTEN by PS is independent of the PS/gamma-secretase activity since impaired gamma-secretase by the gamma-secretase inhibitor treatment or due to nicastrin deficiency has little effect on the protein level of PTEN. Our data suggest an important role for PS in signaling pathways involving PI3K/Akt and PTEN that are crucial for physiological functions and the pathogenesis of multiple diseases.

Transcriptional regulation of APH-1A and increased gamma-secretase cleavage of APP and Notch by HIF-1 and hypoxia.

The proteolytic cleavage of Alzheimer beta-amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/gamma-secretase complex, which consists of presenilins, nicastrin, APH-1, and PEN-2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5'-flanking region of the human APH-1A gene and identified a 271-bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF-1 to the promoter, which affects the promoter activity. Activation of HIF-1 by short-term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH-1A mRNA and protein expression, accompanied by increased secretion of Abeta and decreased APP CTFs formation, indicative of an increase in gamma-secretase activity. NiCl2 treatments had little effect on APP and the other three components of the gamma-secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH-1A expression and the gamma-secretase mediated Abeta and Notch NICD generation are regulated by HIF-1, and the specific control of APH-1A expression may imply physiological functions uniquely assigned to APH-1A.

Tachyplesin activates the classic complement pathway to kill tumor cells.

Tachyplesin is a small, cationic peptide that possesses antitumor properties. However, little is known about its action mechanism. We used phage display to identify a protein that interacted with tachyplesin and isolated a sequence corresponding to the collagen-like domain of C1q, a key component in the complement pathway. Their interaction was subsequently confirmed by both ELISA and affinity precipitation. Tachyplesin seemed to activate the classic complement cascade because it triggered several downstream events, including the cleavage and deposition of C4 and C3 and the formation of C5b-9. When TSU tumor cells were treated with tachyplesin in the presence of serum, activated C4b and C3b could be detected on tumor cells by flow cytometry, Western blotting, and confocal microscopy. However, this effect was blocked when the tumor cells were treated with hyaluronidase or a large excess of hyaluronan, indicating that hyaluronan or related glycosaminoglycans were involved in this process. Treatment of cells with tachyplesin and serum increased in membrane permeability as indicated by the ability of FITC-dextran to enter the cytoplasm. Finally, the combination of tachyplesin and human serum markedly inhibited the proliferation and caused death of TSU cells, and these effects were attenuated if the serum was heat-inactivated or if hyaluronidase was added. Taken together, these observations suggest that tachyplesin binds to both hyaluronan on the cell surface and C1q in the serum and activates the classic complement cascade, which damages the integrity of the membranes of the tumor cells resulting in their death.

Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide.

AIM: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer. METHODS: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry, propidium iodide staining and immunocytochemical analysis. RESULTS: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA. In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G(1) phase significantly increased, and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl-2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax. CONCLUSION: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.

Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells.

AIM: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells. METHODS: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21(WAF1/CIP1) and c-myc genes were examined with in situ hybridization assay. RESULTS: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21(WAF1/CIP1) mRNA increased. CONCLUSION: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21(WAF1/CIP1) mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.

Effects of tachyplesin on proliferation and differentiation of human hepatocellular carcinoma SMMC-7721 cells.

AIM: To investigate the antitumor activities of tachyplesin on human hepatocellular carcinoma (HCC) cells. METHODS: Tachyplesin, isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes, was used to treat the human HCC cell line SMMC-7721. Effects of tachyplesin on the proliferation of SMMC-7721 cells were measured with trypan blue dye exclusion test and HE staining. The morphology and ultrastructure of the cells were examined by light microscopy and transmission electron microscopy, respectively. The activities of gamma-glutamyltransferase (gamma-GT) and tyrosine aminotransferase (TAT) were assayed with biochemical methods. The levels of alpha fetoprotein (alpha-FP), proliferating cell nuclear antigen (PCNA), p21( WAF1/CIP1) and c-myc were examined by immunocytochemistry. RESULTS: After treatment with tachyplesin 3.0 mg/L, the proliferation of SMMC-7721 cells was inhibited significantly, with the cell growth inhibitory rate amounted to 55.57 % and the maximum cell mitotic index declined by 43.68 %. The morphology and ultrastructure underwent restorational alteration. The activity of gamma-GT declined while TAT activity increased obviously, and the levels of alpha-FP and PCNA decreased. Moreover, the expression of p21(WAF1/CIP1) protein was up-regulated and that of c-myc protein was down-regulated. CONCLUSION: Tachyplesin could effectively inhibit the proliferation of hepatocarcinoma cells, reverse the malignant morphological and ultrastructural characteristics, alter the levels of enzymes and antigens, regulate the expression of differentiation-associated oncogene and tumor suppressor gene, and induce hepatocarcinama cell differentiation.

[Tachyplesin-induced differentiation of human hepatocarcinoma cell line SMMC-7721].

BACKGROUND & OBJECTIVE: The study on antitumor activities of marine bioactive substances is an important field in exploiting marine bioactive substances and antitumor drugs. And the induction of tumor cell differentiation is a new strategy for drug therapy of tumors. So the authors used tachyplesin, a marine bioactive substance, to investigate its effects on the differentiation of human hepatocarcinoma SMMC-7721 cells for further studying its antitumor activities and mechanisms. METHODS: Tachyplesin, which was isolated from hemocytes of horseshoe crab (Tachypleus tridentatus), was used to treat human hepatocarcinoma SMMC-7721 cells. Light microscopy and transmission electron microscopy were applied to examine the changes in morphology and ultrastructure of SMMC-7721 cells, respectively. The activities of alkaline phosphatase or the expression of AFP and PCNA were assessed by cytochemistry or immunocytochemistry. RESULTS: In the cells treated with 3.0 micrograms/ml tachyplesin, the morphology and ultrastructure returned to be normal, the activity of alkaline phosphatase was decreased and the levels of AFP and PCNA were downregulated. CONCLUSION: Tachyplesin might effectively reverse the malignant morphology and ultrastructure, change the activity of enzyme and the levels of antigens associated with hepatocarcinoma cell, and induce the differentiation of the human hepatocarcinoma SMMC-7721 cells.

Effects of tachyplesin on the morphology and ultrastructure of human gastric carcinoma cell line BGC-823.

AIM:To investigate the morphological and ultrastructural changes in the human gastric carcinoma cell line BGC-823 after being treated with tachyplesin.METHODS:Tachyplesin was isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes. BGC-823 cells and the cells treated with 2.0mg/L tachyplesin were examined respectively under light microscope, scanning and transmission electron microscope.RESULTS: BGC-823 cells had undergone the restorational alteration in morphology and ultrastructure after tachyplesin treatment. The changes were as follows: the shape of cells was unanimous, the volume enlarged and cells turned to be flat and spread, the nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular, the number of nucleolus reduced and its volume lessened,heter-chromatin decreased while euchromatin increased in nucleus. In the cytoplasm, mitochondria grew in number with consistent structure relatively, Golgi complex turned to be typical and well-developed,rough endoplasmic reticulum increased and polyribosome decreased. The microvilli at cellular surface were rare and the filopodia reduced while lamellipodia increased at the cell edge.CONCLUSION:Tachyplesin could alter the malignant morphological and ultrastructural charact-eristics of human gastric carcinoma cells effectively and have a certain inducing differen-tiation effect on human gastric carcinoma cells.