The effect of the functional ionic group of the viologen derivative on visible-light driven CO2 reduction to formic acid with the system consisting of water-soluble zinc porphyrin and formate dehydrogenase.

The effect of the functional ionic group of 4,4'-bipyridinium salt derivatives (4,4'-BPs) as the electron carrier on the visible-light driven conversion of CO2 to formic acid with the system consisting of water-soluble zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) and formate dehydrogenase (FDH) in the presence of triethanolamine (TEOA) as an electron donor was investigated. 1,1'-Diaminoethyl- (DAV), 1-aminoethyl-1'-methyl- (AMV), 1-carboxymethyl-1'-methyl- (CMV) and 1,1'-dicarboxymethyl-4,4'-bipyridinium salt (DCV) were prepared as the 4,4'-BPs with the functional ionic group. Irradiation of a CO2 saturated buffer solution containing TEOA, ZnTPPS, 4,4'-BP and FDH with visible light irradiation resulted in the production of formic acid. By using 4,4'-BPs with the cationic aminoethyl-group, DAV or AMV as an electron carrier, the effective visible-light driven formic acid production based on the CO2 reduction was observed compared to the 4,4'-BPs with the anionic carboxymethyl-group, CMV or DCV. The formic acid production rate with DAV was approximately 3.2 times higher than that of the system with DCV.

A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol.

BACKGROUND/AIMS: A stress-inducible heat shock protein 70 (HSP70) is one of the best-known endogenous factors protecting cell injury under various pathological conditions. The aim of this study was to examine anti-apoptotic actions of a non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to hydrogen peroxide (H2O2) or ethanol. METHODS: Primary cultures of rat hepatocytes were treated with different concentrations of GGA and exposed to 0.5 mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA fragmentation. RESULTS: Pretreatment with 1 microM GGA for 2 h enhanced nuclear translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA expression, and its accumulation, when the cells were exposed to H202 or ethanol. In association with this accelerated response, GGA suppressed the insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase 3-like proteases, leading to significant inhibition of apoptosis. CONCLUSIONS: GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for enhanced HSP70 induction. Our results suggest that GGA may have a potential benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.

Apoptosis of proliferative cortical tubular epithelia in chronic progressive nephrosis of rats.

To investigate the role of apoptosis in the pathogenesis of rat chronic progressive nephrosis (CPN), the kidney of male F344/DuCrj rats, 19, 59, and 111 weeks of age, was examined histologically. In situ analysis for DNA fragmentation and proliferating cell nuclear antigen (PCNA) was performed simultaneously by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and immunohistochemistry, respectively. CPN was seen in all the kidneys of 59-week-old (n=6) and 111-week-old rats (n=16), correlating significantly (p<0.01) with age. There were apoptotic bodies (ABs) in the single-layered epithelia of dilated tubules (SLD) and the multilayered epithelia (ML) of the cortical tubules. There were no ABs in any of the kidneys of the 19-week-old (n=5) or 59-week-old rats (n=6). Proliferative activity might have been enhanced in the single-layered and flattened epithelia, SLD, and ML of the cortical tubules in the kidneys of the 59-week-old rats (n=6) compared with that in 111-week-old rats (n=8). The correlations between the TUNEL-positive ratio and number of PCNA-positive cells, and age and the CPN grade were significant (p<0.01) exclusively in the ML. Thus, the results suggest that apoptosis occurs in the proliferative ML of rat CPN, and the pathological significance might be the removal of abnormal or excess cells.

Apoptosis and cell proliferation in rat hepatocytes induced by barbiturates.

To examine the effect on cell population in hepatocytes of phenobarbital (PB) and other barbiturates, PB, allobarbital (ALB), barbital sodium (BS) and barbituric acid (BA) were given orally to male rats for 7 consecutive days. Although there was no apparent change in non-promoting BA, hepatomegaly was induced by PB, BS and ALB, which are promoters of hepatocarcinogenesis. In PB- and BS-treated livers, hepatomegaly was attributable to hepatocyte proliferation and enzyme induction. In ALB-treated liver, it was attributable to enzyme induction. The level of cell proliferation was reduced to less than the control values following withdrawal of PB, ALB and BS. It seemed that the degree of suppression of cell proliferation following withdrawal of these compounds correlated to the degree of cell proliferation (PB>BS>ALB) during treatment. In PB-treated liver, apoptosis was induced during treatment, serving to eliminate the excess of hepatocytes. This suggests that short-term administration of PB neither induced suppression of apoptosis nor disturbed homeostasis of hepatocyte populations.

Intraoperative microwave coagulation therapy for large hepatic tumors.

We report new surgical techniques for intraoperative microwave coagulation therapy (IMCT), conducted in three patients with large liver neoplasms with poor liver function or difficult tumor location. Anterolateral thoracotomy was performed for tumors in the right lobe to obtain a good operative field. Four electrode needles were inserted for microwave irradiation, with settings of 60 W, 45 s for coagulation and 1 s for dissociation. Clamping of the hepatoduodenal ligament was performed during IMCT. We began the coagulation at the bottom of the tumor, irradiating the tumor and the surrounding parenchyma to create regional necrosis with a safe margin. With these methods, we treated two women diagnosed with large hepatocellular carcinoma with liver cirrhosis and a man with liver metastasis from rectal cancer. The postoperative course of these patients was uneventful. A marked low-density area was seen in the region of therapy and no enhanced findings were observed on enhanced computed tomography postoperatively. However, in one patient, transcatheter embolization (TAE) was performed 1 month postoperatively because recurrence was noted on the bottom of the tumor. Thus, IMCT destroys the peripheral part of the tumor that may remain viable after TAE, but combination therapy with TAE is preferable, especially when a viable part exists within tumors. IMCT is an active, safe, and nontoxic therapeutic modality for large hepatic tumors, and is particularly applicable in patients with large hepatocellular carcinomas and poor liver function.

Spontaneous pinealoma in a male Crj:CD (SD) IGS rat.

A pinealoma (benign) was found in a 61-week-old male Crj:CD (SD) IGS rat. The neoplasm was located between the cerebral hemispheres and the cerebellum. Histologically, the tumor cells consisted of two cell types: large, pale-staining cells and small dark-staining cells. A fibrovascular stroma divided the tumor cells into incomplete lobules or nest structures. Relatively numerous mitoses were noted in the tumor cells. Ultrastructurally, the tumor cells contained dense-cored vesicles, approximately 120 nm in diameter.

[Secondary hepatic resections in a case of sigmoid colon cancer with multiple liver metastasis (H3) after successful continuous hepatic artery infusion chemotherapy oriented by in vitro chemosensitivity test].

A 43-year-old woman was admitted to our hospital for sigmoid colon cancer with multiple liver metastasis (H3). As preoperative CTAP (CT during arterial portography) examination showed 23 metastatic nodules in the whole liver, hepatic resections were not indicated. Angiographic findings showed that right and left hepatic arteries branched separately from the celiac artery. Sigmoid colon resection with D3 lymph node dissection and catheterization to the right hepatic artery via gastroduodenal artery were undertaken as a first operation. Continuous hepatic artery infusion chemotherapy with MMC, 5-FU oriented by in vitro chemosensitivity test (SDI test: Succinic Dehydrogenase Inhibition test) of primary tumor was performed 7 days after the first operation. After administration of MMC (40 mg) and 5-FU (16,500 mg), metastatic nodules in the right lobe almost disappeared except for the one tumor of S7, but the size and number of the nodules in the left lobes increased. At 10 months after the first operation, the left hepatic lobectomy and extirpation of only one tumor in the right lobe (S7) underwent. This case showed the usefulness of continuous hepatic artery infusion chemotherapy oriented by in vitro chemosensitivity test for multiple liver metastasis from colon cancer.

Suppression of cyclin D1 mRNA expression by the angiogenesis inhibitor TNP-470 (AGM-1470) in vascular endothelial cells.

The effects of TNP-470 on DNA synthesis and the expression of c-myc and cyclin D1 mRNAs were investigated in human umbilical vein endothelial (HUVE) cells synchronized by serum depletion and stimulated with bFGF and serum. DNA synthesis occurring 16 h after stimulation was inhibited when TNP-470 was present from 2 to 6 h after stimulation. C-myc mRNA expression occurring 2 h after stimulation was not inhibited by the addition of TNP-470. Cyclin D1 mRNA expression occurring 6 h after stimulation was suppressed in the presence of TNP-470 from 2 to 6 h after stimulation. On the other hand, cyclin D1 expression was not suppressed in the TNP-470-insensitive human tumor cell line WiDr. These results suggest that the inhibition of HUVE cells by TNP-470 is due to the suppression of cyclin D1 expression in mid G1 phase.

Angiogenesis inhibitor TNP-470 (AGM-1470) potently inhibits the tumor growth of hormone-independent human breast and prostate carcinoma cell lines.

The angiogenesis inhibitor O-(chloroacetyl-carbamoyl)fumagillol (TNP-470) showed antitumor activity in three human cancer xenograft systems. TNP-470 potently inhibited the tumor growth of hormone-independent prostate cancer PC-3 cells and breast cancer MDA-MB-231 cells dose dependently at weekly s.c. doses of 50-200 mg/kg with maximum inhibition of 96 and 88% (tumor growth, 4 and 12% of that in the respective control). In experiments of combination therapy with chemotherapeutic agents, the combination of TNP-470 (100 mg/kg) and cisplatin (5 mg/kg) showed an additive antitumor effect (from treated versus control, 38 and 22% to 5%) against PC-3 carcinoma. 5-Fluorouracil and Adriamycin alone did not significantly inhibit MDA-MB-231 tumor growth (treated versus control, 131 and 64%, respectively). TNP-470 also inhibited tumor growth of WiDr colon cancer; although the inhibition was less marked (treated versus control, 39%) than that observed with the hormone-independent cancers used in this study. In an in vitro study, all the cell lines tested were considerably insensitive to TNP-470 in monolayer cultures (50% inhibitory concentration, approximately 5 micrograms/ml), whereas TNP-470 inhibited the anchorage-independent growth of PC-3 and MDA-MB-231 cells (50% inhibitory concentration, 0.05 and 470 ng/ml, respectively). The inhibitory activity of TNP-470 against anchorage-independent growth correlated well with the in vivo antitumor activity among the cell lines tested. Thus, this inhibitory action may partly contribute to the potent antitumor activity of the angiogenesis inhibitor TNP-470, at least in the case of PC-3 and MDA-MB-231. These results suggest that hormone-independent prostate and breast cancers may be appropriate target diseases for TNP-470 clinical trials.

Inhibition of tumor growth and metastasis of rodent tumors by the angiogenesis inhibitor O-(chloroacetyl-carbamoyl)fumagillol (TNP-470; AGM-1470).

The effect of the potent angiogenesis inhibitor O-(chloroacetyl-carbamoyl)fumagillol (TNP-470), a semisynthetic analogue of fumagillin, on tumor growth and metastasis was studied using rodent tumors. Injection of TNP-470 s.c. inhibited tumor growth in a dose-dependent manner, and the tumor sizes of B16BL6 melanoma, M5076 reticulum cell sarcoma, Lewis lung carcinoma, and Walker 256 carcinoma were maximally reduced to 16, 10, 17, and 4% of that in the respective control. The activity of TNP-470 upon i.v. injection was slightly weaker than that following s.c. injection. This tendency was observed for all the tumors tested. Injection i.v. (infusion) of TNP-470 increased the life span of Walker 256 carcinoma-bearing rats by 183% over the control, while bolus i.v. injection increased the life span by only 47%. TNP-470 reduced the number of pulmonary metastatic foci of i.v. inoculated B16BL6 melanoma in a dose-dependent manner, and the number of metastatic foci was reduced to 10% of that in the control by treatment with TNP-470 at 60 mg/kg, 3 times/week. The mean survival time of B16BL6 tumor-bearing mice treated with TNP-470 using this regimen was extended by 56% over that of control mice. TNP-470 at 10 mg/kg every day also reduced the number of metastatic foci of M5076 sarcoma in the liver after resection of the tumor from the primary site. Adriamycin at the same dose only slightly reduced the number of metastatic foci, even though TNP-470 and Adriamycin showed roughly equal inhibitory activity against M5076 sarcoma growth. TNP-470 extended the mean survival time of M5076 tumor-bearing mice by more than 100% over that of control mice at 30 mg/kg every 3 days, while Adriamycin extended mean survival times by maximally 20% at 10 mg/kg. These results show that the angiogenesis inhibitor TNP-470 has strong inhibitory activities against in vivo growth and metastasis of a wide variety of tumors.

Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.

Previously we showed that motility-related protein (MRP-1) is an antigen recognized by monoclonal antibody (mAb) M31-15 inhibiting cell motility and that the sequence of MRP-1 coincides with that of CD9. In the present study, plasmid was constructed in which human MRP-1/CD9 cDNA is expressed under the control of the Abelson murine leukemia virus promoter sequence. The expression plasmid for MRP-1/CD9 was introduced into Chinese hamster ovary cells, human lung adenocarcinoma cell line MAC10 (MRP-1 positive), and human myeloma cell line ARH77 (MRP-1 negative). All of the MRP-1/CD9 (over)expressing clones obtained from these transfected cells showed suppressed cell motility (penetration and phagokinetic track assays) depending on the degree of expression of MRP-1/CD9. Overexpression of MRP-1/CD9 by MAC10 cells resulted in the suppression of cell motility (maximally 73%) associated with considerable inhibition of the cell growth (maximally 48%). However, the inhibition of the growth of MAC10 cells by mAb M31-15 was < 17% at an antibody concentration of 1-5 micrograms/ml, which inhibits cell motility by > 90%. These results suggest that MRP-1/CD9 directly regulates cell motility and may also affect cell growth. Effects on metastasis by the expression of MRP-1 CD9 were investigated with mouse melanoma BL6 cells-BALB/c nu/nu mouse system. Metastatic potential of all transformants expressing MRP-1/CD9 was lower than that of parent BL6 cells.

Involvement of CD8+ T cells in delayed-type hypersensitivity responses against bovine leukemia virus (BLV) induced in sheep vaccinated with recombinant vaccinia virus expressing BLV envelope glycoprotein.

Delayed-type hypersensitivity responses against bovine leukemia virus (BLV) envelope glycoprotein (gp60) were induced in the skin of sheep vaccinated with recombinant vaccinia virus (RVV) expressing BLV glycoprotein. The lesions were characterized by marked infiltration of lymphocytes, slight migration of neutrophils, eosinophils, and macrophages in the dermis to hypodermis, and partial intercellular edema in the reticular layer. Immunohistochemical analysis with monoclonal antibodies demonstrated that the lymphocytic infiltrates consisted mainly of CD8+ T cells (53.7-55.8% at 48 hours post-challenge of BLV), CD4+ T cells (24.7-26.7%), and B cells (11.5-16.9%). The role of CD4+ and CD8+ T cells in suppressing BLV growth in RVV-vaccinated animals is discussed.

Therapeutic effect of ansamitocin targeted to tumor by a bispecific monoclonal antibody.

We have constructed a murine hybrid hybridoma that secretes a bispecific monoclonal antibody (mAb) by fusing a hybridoma secreting an anti-ansamitocins mAb with a hybridoma secreting an anti-human transferrin receptor (TfR) mAb that binds to human A431 epidermoid carcinoma cells. The bispecific mAb, reactive to both ansamitocins and TfR, was purified by a combination of hydrophobic column chromatography and hydroxyapatite high-performance liquid chromatography, and evaluated in in vivo experiments using human tumor cell-implanted nude mice. Ansamitocin P-3 targeted through one of the antigen combining sites of the bispecific mAb was potentially more effective in suppressing the growth of established A431 tumor xenografts implanted on nude mice than unconjugated ansamitocin P-3 or the immunoconjugate of ansamitocin P-3 and monospecific anti-ansamitocins antibody, and the targeted ansamitocin P-3 finally eradicated the tumor mass. The bispecific mAb also played an important role in reducing such undesirable side-effects of ansamitocin P-3 as the loss of body weight, the damage to liver functions and the decrease in the number of white blood cells.

Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility.

A murine monoclonal antibody (M31-15) was identified using the penetration-inhibiting assay of a human lung adenocarcinoma cell line (MAC10) and remarkably inhibited the phagokinetic tract motility of various cancer cell lines. The antigen, motility-related protein (MRP-1), recognized by M31-15, was 25- and 28-kD proteins, and M31-15 was used to isolate a cDNA clone from a human breast carcinoma cDNA library. Sequence analysis revealed that MRP-1 had strong similarity with a B cell surface antigen (CD37), a melanoma-associated antigen (ME491), the target of an antiproliferative antibody (TAPA-1), a human tumor-associated antigen (CO-029), and the Sm23 antigen of the trematode parasite Schistosoma mansoni.

Purification and characterization of a recombinant human IgE Fc epsilon fragment lacking the C4 domain.

Complementary DNA of human IgE Fc epsilon fragment (residues 226-480) lacking the C epsilon 4 domain was expressed in Escherichia coli and the product was purified by immunoaffinity chromatography on a monoclonal antibody (E12 0.02)-Affi-Gel 10 column. About 1.8 mg of an apparent dimer and 5.9 mg of a monomer were obtained from 65 g E. coli cells with 9.3% recovery. The purified products were found to lack more than half of the COOH-terminal portion of the C epsilon 3 domain. The apparent dimer showed high immunological specific activity (3.6 x 10(6) U/mg protein) comparable to that of natural human IgE when measured by commercial human IgE determination kits.

Purification and characterization of recombinant human IgE Fc epsilon fragment produced in mouse L cells.

A human IgE Fc epsilon fragment was isolated from the supernatant of the culture fluid of a recombinant mouse L cell line, L-IS11IgE-9. The purification procedure consisted of salting out from the supernatant with ammonium sulfate, immunoaffinity chromatography on a monoclonal antibody (E235I63)-Affi Gel 10 column, and gel filtration chromatography on a Sephacryl S-200 column. The final preparation represented a 5825-fold purification from the original culture fluid with a 25% recovery and about 3.1 mg of Fc epsilon fragment was obtained from 201 of culture fluid. The sp. act. of the purified preparation measured by the use of commercial human IgE determination kits was 0.93 x 10(6) units/mg protein. The purified preparation was homogeneous as judged by the end group analyses. The amino acid composition of the preparation coincided with that deduced from DNA sequence. The mol. wt of our preparation was about 110,000 under non-reducing conditions and 55,000 under reducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results showed that our preparation was a dimeric form having high reactivity against anti-human IgE antibodies.

Differential purification by immunoaffinity chromatography of two carboxy-terminal portion-deleted derivatives of recombinant human interferon-gamma from Escherichia coli.

Two lower-molecular-weight derivatives of recombinant human interferon-gamma (rIFN-gamma) were purified concurrently from a lysozyme-EDTA extract of Escherichia coli cells by immunoaffinity chromatography using a monoclonal antibody (MAb) against a synthetic carboxy-terminal peptide (Lys-131-Gln-146). The two derivatives, 15K rIFN-gamma and 17K rIFN-gamma, were regarded to have been generated at the extraction step. They were successfully separated from each other by using another MAb against the same synthetic peptide with higher binding affinity than the first. The results of protein-chemical analyses indicate that 15K rIFN-gamma and 17K rIFN-gamma lack 15 (Arg 132-Gln-146) and 4 (Arg-143-Gln-146) carboxy-terminal amino acid residues, respectively. All the data suggest that the two derivatives form a noncovalent dimer and that 15K rIFN-gamma binds indirectly to the MAb column via 17K rIFN-gamma.

Thermally induced disulfide bond exchanges in human IgE.

The thermally induced changes in human IgE was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the IgE (1 mg/ml) was heated at 56 degrees C for several hours in the absence of sodium dodecyl sulfate, it gave aggregated forms that could be reduced completely to heavy and light chains with dithiothreitol. On the other hand, in the presence of sodium dodecyl sulfate (0.1%), the IgE was changed by heating to five components with small mol. wts dependent on temperature and pH. This phenomenon was observed also in mouse monoclonal IgG1, but the degree of change was considerably lower than that of human IgE. The five components obtained from the heat-treated IgE (LHHL) were identified as LHHL, HL, H, LL and L. These results show that the thermally induced changes in human IgE are dependent on the exchange of its intra- and inter-disulfide bonds.

Purification and characterization of IgE produced by human myeloma cell line, U266.

Human IgE was isolated for the first time from the supernatant of the culture fluid of a human myeloma cell line, U266. The purification procedure consisted of salting out from the supernatant with ammonium sulfate, affinity chromatography on a lysine-Sepharose 4B column, ion exchange chromatography on a DEAE-Sephacel column, gel filtration and recycling chromatography on a Sephacryl S-300 column and removal of bovine proteins on an anti-bovine serum rabbit IgG-Sepharose 4B column. One hundred and twenty eight milligrams of IgE was recovered from 461 of culture fluid. The purification was extremely simplified by the introduction of immunoaffinity chromatography using the monoclonal antibody prepared by immunizing a mouse with an IgE preparation obtained by the above method: about 3.3 mg was recovered from 960 ml of culture fluid. The purified preparation was homogeneous as judged by the double-immunodiffusion test and end group analysis. The amino acid and carbohydrate compositions of the preparation coincided with those reported on other preparations obtained from the sera of myeloma patients. Our preparation, however, showed two bands with apparent mol. wts of 240,000 and 230,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When it was reduced with dithiothreitol and analyzed by electrophoresis, it gave two heavy chains and one light chain with apparent mol. wts of 80,000 and 76,000, and 28,000, respectively. On the other hand, the IgE molecule that was synthesized and secreted into the medium in the presence of tunicamycin (0.5 microgram/ml) gave only one heavy chain and one light chain with apparent mol. wts of 62,000 and 28,000, respectively. These results demonstrated that the two IgE molecular species contained in our preparation differed from each other in the carbohydrate moiety in their heavy chains.