Thrombin cleaves recombinant soluble thrombomodulin into a lectin-like domain fragment and a fragment with protein C-activating cofactor activity.

Thrombomodulin (TM) is a transmembrane protein that plays an important role in regulating the coagulation system by acting as a cofactor for thrombin in protein C activation. Additionally, TM is involved in inflammation. Previous studies have shown that soluble fragments of TM of varying sizes, which are derived from membrane-bound TM, are present in plasma and urine. Soluble fragments of TM are speculated to exhibit biological activity. Among these, a lectin-like domain fragment (TMD1) is of particular importance. Recombinant TMD1 has previously been shown to attenuate lipopolysaccharide-induced inflammation. Here, we report that thrombin cleaves recombinant soluble TM, which is used for the treatment of disseminated intravascular coagulation associated with sepsis, into TMD1 and a fragment comprising the C-terminal portion of TM (TMD23), the latter of which retains the cofactor activity for activating protein C. Our findings suggest that thrombin not only activates protein C on membrane-bound TM but may also cleave TM to generate TMD1.

Structural analyses of a hemolytic compound found in an extract of Hypsizygus marmoreus fruiting bodies at a low pH.

The current study determined the structure of a hemolytic compound found in an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus when its pH was lowered. The hemolytic compound was purified using the modified Bligh and Dyer method followed by chromatography using reversed phase and silica gel columns. Structural analyses of the purified hemolytic compound were performed using NMR and ESI-MS. The deduced structure indicated a trans,trans-5,8-docosadienoic acid calcium salt. Although numerous proteinous hemolysins from various mushrooms have been described, the current study is the first to report on a low-molecular-weight hemolytic compound derived from an H. marmoreus extract.

pH-Dependent exhibition of hemolytic activity by an extract of Hypsizygus marmoreus fruiting bodies.

The current study found that an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus exhibited hemolytic activity against sheep red blood cells when its pH was lowered. Although hemolytic activity was not detected when an extract had a neutral pH, an extract with a low pH exhibited potent hemolytic activity. The maximal hemolytic activity was exhibited by an extract with a pH of 5.5. A heat-treated extract did not exhibit hemolytic activity before its pH was lowered, and that activity was inhibited in the presence of PMSF and EDTA. The turbidity of the extract increased during lowering of its pH, and the precipitate fraction exhibited hemolytic activity. Fractionation by a modified Bligh and Dyer method and TLC analyses suggested that a hemolytic compound in the extract might be a type of lipid. These results suggest that a hemolytic lipid-like compound in an extract of H. marmoreus fruiting bodies may be released by a non-active precursor substance(s) through metalloenzyme(s) while the extract has a low pH.

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain.

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle-aged Tet-mev-1 mice with the SDHCV69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle-aged Tet-mev-1 mice overproduced MitoSOX Red-detectable mitochondrial ROS compared to age-matched wild-type C57BL/6J mice, only young adult Tet-mev-1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn- and Cu/Zn-SODs) activities to eliminate the MitoSOX Red-detectable mitochondrial ROS. In contrast, middle-aged Tet-mev-1 mice accumulated both MitoSOX Red-detectable mitochondrial ROS and CM-H2 DCFDA-detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle-aged Tet-mev-1 mice relative to age-matched wild-type C57BL/6J mice. Furthermore, only middle-aged Tet-mev-1 mice showed JNK/SAPK activation and Ca2+ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100ß in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age-dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.

Toad skin extract cinobufatini inhibits migration of human breast carcinoma MDA-MB-231 cells into a model stromal tissue.

Toad skin extract cinobufatini study has been focused on anticancer activity, especially apoptosis-inducing activity by bufosteroids. The present study examined effect of the toad skin extract on cancer cell migration into model stromal tissues. Human breast carcinoma cell line MDA-MB-231 was incubated in the presence or absence of toad skin extract on a surface of reconstituted type I collagen gel as a model stromal tissue allowing the cells to migrate into the gel. Frozen sections were microscopically observed after azan staining. Data showed a decrease of cell number in a microscopic field and shortening of cell migration into the model stromal tissue in a dose dependent manner. This suggests that toad skin extract may possess migration-preventing activity in addition to cell toxicity such as apoptosis-inducing activity. The multifaceted effects including apoptosis-inducing and cancer cell migration-preventing activities would improve usefulness of toad skin extract cinobufatini as an anticancer medicine.

Susceptibility to proteases of anti-Tn-antigen MLS128 binding glycoproteins expressed in human colon cancer cells.

Anti-Tn antigen MLS128 monoclonal antibody was produced two decades ago by immunizing mice with "cancerous antigens" derived from LS180 colon cancer cells. Previous studies demonstrated that MLS128 bound to 110 kDa glycoprotein (GP) in colon cancer cells, thereby inhibiting cell growth. Extensive attempts have been made towards understanding the inhibitory action of MLS128 on colon cancer cell growth and solving the primary structure of 110 kDa GP. Since limited proteolysis of 110 kDa GP was observed in microdomain fractions that had been kept frozen for several years, susceptibility of 110 kDa GP to trypsin and other proteases as well as N-glycosidase F has been investigated. Furthermore, 110 kDa GP expression was examined in colon cancer cells independently cultured in Akiyama laboratory. In summary, 110 kDa GP contains N-glycans. It does not contain inter-disulfide bonds but appears to have intra-disulfides. It must contain multiple cleavage sites for trypsin and thermolysin since these proteases digested 110 kDa GP to MLS128-undetectable small fragments. It seems to contain cleavage sites for cathepsin D which could cause limited digestion. LS180 cells derived from Akiyama laboratory produced a limited proteolysis product-like 75 kDa GP. This study provides a structural basis for developing cancer diagnostics and therapeutics.

An aqueous extract from toad skin prevents gelatinase activities derived from fetal serum albumin and serum-free culture medium of human breast carcinoma MDA-MB-231 cells.

An aqueous extract from toad skin, cinobufacini, has been known to possess anticancer ability. The present study examined effect of toad skin extract on activity of gelatinases including matrix metalloproteinases-2 and -9 which play an important role in invasion of carcinoma cells. Gelatinase activities derived from fetal serum albumin and culture medium of human breast carcinoma cell line MDA-MB-231 were significantly prevented in the presence of toad skin extract. The inhibitory activity was found in water-soluble fraction of the extract prepared by the Bligh & Dyer method but not in CHCl(3)-soluble lipid fraction. These results suggest that an aqueous extract from toad skin contains a water-soluble substance possessing a potent ability to prevent gelatinase activity. In conclusion, the water-soluble substance in toad skin extract cinobufacini may be able to regulate cancer cell migration accelerated by matrix metalloproteinases.

Migration of breast cancer cells into reconstituted type I collagen gels assessed via a combination of frozen sectioning and azan staining.

This study sought to devise a way to assess the infiltration of cancer cells in model stromal tissues. Human breast carcinoma MDA-MB-231 cells were loaded on the surface of a type I collagen gel in the well of 8-well chamber slide and allowed to migrate into the gel. The gel was then subjected to frozen sectioning and staining. Azan staining facilitated satisfactory microscopic observation of cancer cells migrating into the collagen gel. Cell migration was promoted by the presence of fetal calf serum in the gel. In contrast, the proportion of cells remaining on the gel surface increased in the presence of galardin, a matrix metalloproteinase inhibitor. Moreover, the distance of cell migration from the gel surface was significantly shorter depending on the concentration of galardin. Observation of cancer cell migration into reconstituted type I collagen gel with a combination of frozen sectioning and azan staining is a useful way to assess the ability of individual cancer cells to migrate and to evaluate how effectively pharmaceuticals inhibit the first step of invasion.

Clinicopathological utility of sialoglycoconjugates in diagnosing and treating colorectal cancer.

Aberrant expression of glycoconjugates occurs during malignant transformation of cancer cells. Overexpression of sialoglycoconjugates in particular may play an important role in the progression, i.e., invasion or metastasis, of cancer. Various types of sialoglycoconjugates have been investigated to clarify their biological significance and clinical utility in diagnosing and treating colorectal cancer. This review focuses specifically on expression of mucin (MUC) 1 and it suggests that MUC1 with the specific structure of a sialo-oligosaccharide has biological significance in determining the metastatic potential of colorectal cancer cells and clinicopathological utility in evaluating the effectiveness of treatments and the prognosis for patients with colorectal cancer. Further studies are expected to contribute to the expanded use of cancer-associated sialoglycoconjugates in cancer diagnosis and therapy.

Characterization of three different single chain antibodies recognizing non-reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system.

We previously isolated phage antibodies from a phage library displaying human single chain antibodies (scFvs) by screening with a mannotriose (Man3)-bearing lipid. Of four independent scFv genes originally characterized, 5A3 gene products were purified as fusion proteins such as a scFv-human IgG1 Fc form, but stable clones secreting 1A4 and 1G4 scFv-Fc proteins had never been established. Thus, bacterial expression systems were used to purify 1A4 and 1G4 scFv gene products as soluble forms. Purification of 1A4 and 1G4 scFv proteins from inclusion bodies was also carried out together with purification of 5A3 scFv protein in order to compare their Man3-binding abilities. The present studies demonstrated that 1A4 and 1G4 scFv proteins have a higher affinity for Man3 than 5A3 scFv protein, which may determine whether scFv-Fc proteins expressed in mammalian cells are retained in the ER or secreted. Furthermore, the inhibitory effects of anti-Man3 1G4 scFv and anti-Tn antigen scFv proteins on MCF-7 cell growth were evaluated. Despite the fact that no obvious difference was detected in cell growth, microscopic observations revealed inhibition of foci formation in cells grown in the presence of the anti-carbohydrate scFv proteins. This finding provides a basis for the development of cancer therapeutics.

Sustained aberrant localization of KL-6 mucin and beta-catenin at the invasion front of human gastric cancer cells.

AIMS: This study sought to analyze the clinicopathological significance of KL-6 mucin, a type of MUC1, in gastric cancer and its relationship to ß-catenin. PATIENTS AND METHODS: Subcellular localization of KL-6 mucin and ß-catenin in gastric cancer tissues (n = 96) was detected immunohistochemically and its clinicopathological significance was evaluated. RESULTS: Samples with localization of KL-6 mucin in the surrounding membrane and/or cytoplasm of cancer cells at the invasion front (n = 50, 52%) had a higher incidence of deeper invasion (p < 0.0001), lymphatic vessel invasion (p = 0.0003), venous invasion (p < 0.0001), lymph node metastasis (p < 0.0001) and an advanced TNM stage (p = 0.0005). Furthermore, this localization of KL-6 mucin was associated with accumulated nuclear localization of ß-catenin. CONCLUSION: In concert with aberrant localization of ß-catenin, sustained localization of KL-6 mucin in the surrounding membrane and/or cytoplasm of cancer cells at the invasion front has a significant role in cancer progression.

Antitumor activity of extracts and compounds from the skin of the toad Bufo bufo gargarizans Cantor.

The skin of the toad Bufo bufo gargarizans Cantor is known to be rich in bufadienolides, peptides and alkaloids. It has been found to be a source of some extracts and biologically active compounds with antitumor activity. Cinobufacini (Huachansu), a Chinese medicine prepared from the dried toad skin, has been widely used in clinical therapy for various cancers in China. Bufadienolides, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are the major active compounds derived from the toad skin. They are the maker biologically active compounds of cinobufagin while the antitumor activity of cinobufagin may be due to this kind of components. Experimental research has suggested that cinobufacini and its active compounds (e.g. bufalin and cinobufagin) exhibit significant antitumor activity, including inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response. Clinical data have indicated that cinobufacini may have effective anticancer activity with low toxicity and few side effects. Data to date suggest it may also enhance quality of life for patients with cancer. Thus, this review briefly summarizes recent studies on the anticancer activity of cinobufacini and some of its active compounds from the skin of the toad Bufo bufo gargarizans Cantor. This might provide additional evidence for further study of the extracts and active compounds from the toad skin in cancer treatment.

Production of anti-carbohydrate antibodies by phage display technologies: potential impairment of cell growth as a result of endogenous expression.

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG(1) Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253-262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263-270). Similarly, anti-Le(x) phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le(x))-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.

Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells.

AIM OF THE STUDY: Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402. MATERIALS AND METHODS: The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis. RESULTS: Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP). CONCLUSION: The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.

Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments.

The Tn-antigen, GalNAcalpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.

Preparation of asialo-agalacto-glycophorin A for screening of anti-Tn antibodies.

Oncogenic antigens such as Tn-antigen (GalNAcα-Ser/Thr) are involved in metastatic processes and are associated with a poor prognosis, thus representing excellent targets for cancer intervention. Available anti-Tn antibodies which can be applied for therapeutics or diagnostics are severely limited mostly because the Tn-antigen epitope by itself is too small to be antigenic in addition to the fact that many carbohydrates are self-antigens. To characterize anti-Tn monoclonal antibodies as well as to perform panning and screening for isolation of anti-Tn single chain variable fragments from phage-display libraries, a large quantity of inexpensive Tn-antigens are needed. In this study, thus, glycophorin A which is a highly glycosylated sialoglycoprotein with approximately 12 O-glycans was sequentially treated with sialidase and ß-galactosidase to remove sialic acid and galactose residues. The resulted product was shown to be an asialo-agalacto-glycophorin A which is reactive to an anti-Tn-antigen antibody. The simple preparation procedures described here would greatly help production and characterization of potentially valuable anti-Tn-antigen antibodies, which can be readily developed for cancer therapeutics and diagnostics.

KL-6 mucin in metastatic liver cancer tissues from primary colorectal carcinoma.

BACKGROUND/AIMS: KL-6 mucin is MUC1 bearing sialylated carbohydrate epitopes recognized by KL-6 antibody. This study aimed to assess subcellular localization of KL-6 mucin in liver metastatic tissues from colorectal carcinoma and hepatocellular carcinoma tissues. METHODOLOGY: Tissue samples were collected from 56 patients with liver metastasis of colorectal carcinoma and 92 patients with hepatocellular carcinoma who underwent a hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody. RESULTS: All 56 cases with metastatic liver cancer showed positive staining for KL-6 mucin in cancer tissues but not in non-cancerous tissues. All staining was observed in the circumferential membrane and/or cytoplasm of the cancer cells. In contrast, no staining for KL-6 mucin was observed in either cancerous or non-cancerous tissues of the 92 patients with hepatocellular carcinoma. CONCLUSIONS: KL-6 mucin may be an indicator for liver metastasis of colorectal carcinoma. Additionally, detection of KL-6 mucin may be helpful in distinguishing hepatocellular carcinoma from metastatic liver cancer.

A novel measurement method for activation of the lectin complement pathway via both mannose-binding lectin (MBL) and L-ficolin.

Mannose-binding lectin (MBL), L-ficolin and H-ficolin are human serum lectins, all of which form complexes with MBL-associated serine proteases (MASP). The lectin-MASP complexes bind to the surface of microbes, leading to activation of the lectin pathway of complement. Enzyme-linked immunosorbent assays (ELISA) of the lectin pathway activity reported so far determined the activity via either MBL or L-ficolin, but an assay of activity via plural host defense lectins has not been established. To measure the lectin pathway activation mediated by plural lectins simultaneously, we developed an ELISA system in which N-acetylglucosamine-pentamer conjugated to dipalmitoylphosphatidylethanolamine (GN5-DPPE) was employed as a ligand for the lectins. In our ELISA system, both purified MBL and L-ficolin isolated from serum diluted in a buffer containing high ionic NaCl bound to GN5-DPPE and activated C4. Purified H-ficolin was not capable of binding to GN5-DPPE. MBL and L-ficolin in MBL-sufficient serum also bound to GN5-DPPE and activated C4. Mannose and N-acetylgalactosamine inhibited binding of MBL and L-ficolin to GN5-DPPE, respectively. MBL-deficient serum that had been depleted of L-ficolin did not exhibit C4 activation, but addition of both or either purified MBL and/or L-ficolin to the serum restored the activation in a dose-dependent manner. Thus, C4 cleaving activity could be evaluated with the co-existence of MBL and L-ficolin in vitro. In conclusion, we propose a novel method using GN5-DPPE for investigating the MBL- and L-ficolin-dependent lectin pathway and anticipate that this method will be useful in innate immunity and clinical research.

Factor G utilizes a carbohydrate-binding cleft that is conserved between horseshoe crab and bacteria for the recognition of beta-1,3-D-glucans.

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.