Immunogenicity of human melanoma-associated antigens defined by murine monoclonal antibodies in allogeneic and xenogeneic hosts.

The immunogenicity in patients with melanoma, in monkeys, and in rabbits of four human melanoma-associated antigens (MAA) defined by murine monoclonal antibodies was investigated. The latter included the high molecular weight MAA and the (Mr 115,000, 100,000, and 95,000-150,000 MAA. To this end sera from patients with melanoma, from monkeys, and from rabbits immunized with cultured human melanoma cells were tested for their ability to inhibit the binding to cultured human melanoma cells of radiolabeled anti-Mr 95,000-150,000 MAA monoclonal antibody (MoAb) 140.72, anti-high molecular weight MAA MoAb 225.28, anti-Mr 115,000 MAA MoAb 345.134, and anti-Mr 100,000 MAA MoAb 376.96. None of the sera from patients with melanoma significantly inhibited the reactivity of any of the anti-MAA monoclonal antibodies with melanoma cells. Of the sera from the six monkeys immunized with human melanoma cells, two sera significantly inhibited the reactivity with cultured human melanoma cells of both MoAb 345.134 and 376.96, one serum inhibited only that of MoAb 345.134, and the remaining three sera did not inhibit any of the four anti-human MAA monoclonal antibodies. Sera from six of the seven rabbits immunized with cultured human melanoma cells inhibited the binding to melanoma cells of at least one of the four anti-human MAA monoclonal antibodies while the serum from one rabbit immunized with a melanoma cell extract had no effect. Marked differences were found among the individual rabbit sera in their ability to inhibit the binding of the four anti-human MAA monoclonal antibodies. Sequential immunoprecipitation experiments corroborated the serological findings obtained with one of the two rabbit antisera tested. These results suggest that the immunogenicity of human MAA in mice may be different from that in patients with melanoma and in other animal species.

A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the QcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex.

Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its E(m, 7) was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3'-downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe-S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b(6) show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b(6) and green sulfur-low G+C Gram-positive bacteria.

Chemistry and inhibitory activity of long chain fatty acid oxidation of emeriamine and its analogues.

Emericedins A, B, and C, new betaines having inhibitory activity of long chain fatty acid oxidation, were isolated from the culture broth of Emericella quadrilineata IFO 5859. Their structures were determined by spectroscopic analyses as (R)-3-(acylamino)-4-(trimethylammonio)butyrate (acyl: A, acetyl; B, propionyl; C, n-butyryl). Structural confirmation and assignment of absolute configuration were made by chemical synthesis from L-asparagine. Deacylation of emericedin gave a potent derivative, (R)-3-amino-4-(trimethylammonio)butyrate, designated as emeriamine. In order to study the structure-activity relations, various analogues of emeriamine, including a stereoisomer, were prepared. Among them, N-palmitoyl and N-myristoyl derivatives showed much stronger inhibition of fatty acid oxidation than emeriamine.

Inhibition of prolyl hydroxylase activity and collagen biosynthesis by the anthraquinone glycoside, P-1894B, an inhibitor produced by Streptomyces albogriseolus.

P-1894B, a potent prolyl hydroxylase inhibitor produced by Streptomyces albogriseolus subsp. No. 1894, inhibited about 50% of the activity of purified chick embryo prolyl hydroxylase at a concentration of 2.2 x 10(-6) M. The inhibition was noncompetitive with respect to (Pro-Pro-Gly)5 with a Ki of 1.8 x 10(-6)M. When excess amounts of ferrous ions or ascorbate were added to the reaction mixture, the inhibition was slightly reversed. P-1894B at a dose of 0.15 mg/kg reduced the hydroxylation of peptidyl proline and caused a significant inhibition of collagen biosynthesis in the uterus of the immature rat stimulated by the administration of estradiol-17 beta.

Levels of telomerase catalytic subunit mRNA as a predictor of potential malignancy.

This study investigated the relationship between the levels of human telomerase reverse transcriptase (hTERT) mRNA and that of telomerase activity in hepatocellular carcinoma (HCC). A significant correlation between hTERT mRNA expression and telomerase activity by transfecting the gene encoding hTERT into telomerase-negative human fibroblast cells has clearly been demonstrated. However, the relationship between levels of telomerase activity and that of hTERT mRNA has yet to be elucidated. In this study, the levels of hTERT mRNA were analyzed in 24 HCC patients by real-time PCR. And the intensity of telomerase activity was analyzed by fluorescence-based TRAP method. The difference of hTERT mRNA level was highly significant between tumor tissues and non-cancerous liver tissues. And there were significant correlations between the levels of hTERT mRNA and that of telomerase activity (r=0.751) in tumor tissues. We observed a strong correlation between levels of hTERT mRNA and that of telomerase activity in HCC. Our results suggest that the levels of hTERT mRNA would be useful in genetic diagnostic tests, instead of telomerase activity, to screen at-risk patients of HCC in human liver tissues.

A new method for preparation of an antiserum to penicillin and its application for novel enzyme immunoassay of penicillin.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.

Telomerase activity in hepatocellular carcinoma as a predictor of postoperative recurrence.

Telomerase is a ribonucleoprotein that stabilizes telomeres and allows unlimited cell division. It has been reported that most cancer cells evince reactivated telomerase. We examined telomerase activity in 29 patients with hepatocellular carcinoma (HCC) by a polymerase chain reaction-based semiquantitative assay. Of 24 HCCs, telomerase activity was positive in 23 (95.8%), of which 16 showed strong activity. In 11 well differentiated HCCs, telomerase activity was strong in 5, weak in 5, and undetected in 1 and in 13 moderately differentiated HCCs, it was strong in 11 and weak in 2. Five of 6 HCCs less than 2 cm in diameter expressed strong telomerase activity, while weak telomerase activity was detected in 7 of 19 (36.8%) resected noncancerous liver tissues from the HCC patients. Five of these 7 patients (71%) manifested recurrence within 6 months after surgery. The recurrence rate in these patients whose noncancerous liver tissue was positive for telomerase activity was significantly higher than that in patients in whom it was negative (P = 0.017). These results suggest that the presence of telomerase activity may be a useful diagnostic marker of HCC, regardless of tumor size, and that its detection in resected noncancerous liver tissues may serve as a useful predictor of postoperative recurrence.

Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis.

A case of pancreatic serous cystadenoma obstructing the distal pancreatic duct.

We present a case of resected serous cystadenoma of the pancreas inducing marked dilatation of the main distal pancreatic duct. A 68-year-old woman, previously diagnosed with chronic pancreatitis, presented with upper abdominal pain. Abdominal US revealed a highly echoic mass in the pancreas. A CT scan disclosed a low density mass in the pancreas and dilatation of the main peripheral pancreatic duct. The mass demonstrated homogeneous and high signal intensity on T2-weighted magnetic resonance imaging (MRI). Selective abdominal arteriography showed the mass strained by the celiac artery. The tumor markers were CEA (2.4 ng/ml) and CA19-9 (6.1 U/ml). After the diagnosis of serous cystadenoma of the pancreas, the patient underwent distal pancreatectomy and splenectomy. The tumor (2.5 cm in diameter) consisted of grayish-white nodules and occupied the body of the pancreas. The tail of the pancreas was atrophic. Histopathological examination of the specimen showed a multilocular lesion containing numerous cysts with the inner surfaces evenly lined by one layer of cuboid or flat epithelial cells which stained positive for periodic acid-Schiff (PAS), evidencing serous cystadenoma. The patient is doing quite well one and a half years after the operation.

A hepatocellular carcinoma with lymph node metastasis and invasion into the gallbladder: preoperative difficulty ruling out a gallbladder carcinoma.

We present a case of resected hepatocellular carcinoma (HCC) which invaded the gallbladder with a metastasis to a lymph node. It was extremely difficult to make a differential diagnosis between HCC and gallbladder cancer preoperatively. A 68-year old man was admitted to hospital with complaint of a fever. Ultrasonography (US) and CT scan showed a mass, growing invasively from the gallbladder bed of the liver (S4) to the lumen of the gallbladder. A selective arteriography showed the mass stained by the cholecystic artery, internal branch of the left hepatic artery, and frontal branch of the right hepatic artery. Endoscopic retrograde cholangiopancreatography (ERCP) showed the non-visualized gallbladder, a constriction of the common hepatic duct with suspicion of metastatic lymph nodes in the hepatoduodenal ligament. The tumor markers were: alpha-fet-protein 13175 ng/ml, PIVKA-II 26200 mAU/ml and CA19-9 0.0 U/ml. Both HBs antigen and HCV antibody were negative. We performed cholecystectomy with en-block resection of the anterior and middle inferior segment of the liver, the common bile duct and a part of the transverse colon, with dissection of the lymph nodes. The tumor, 8 cm in diameter, was brown colored without a capsule, growing diffusely in the liver, to the inside of the gallbladder and the transverse colon. Histopathological inspection of the specimen revealed moderately differentiated hepatocellular carcinoma with a metastatic lymph node along the common hepatic artery. TNM classification was IVB phase [T3,N0,M1 (LYM,OTH)]. There are only 3 previous cases of HCC reported with invasion into the gallbladder. At most 2.2% of the resected cases of HCC had metastatic lymph nodes at resection, while it was as high as 20-50% of the autopsy cases. Operation on such an invasive HCC case should consider lymph node metastasis.

Emeriamine, an antidiabetic beta-aminobetaine derived from a novel fungal metabolite.

Emeriamine [(R)-3-amino-4-trimethylaminobutyric acid], derived from a novel fungal metabolite "emericedin" [(R)-3-acetylamino-4-trimethylaminobutyric acid], was proved to be a strong and specific inhibitor of carnitine-dependent oxidation of long chain fatty acid (IC50; 3.2 X 10(-6)M) and its main inhibition site was shown to be carnitine palmitoyltransferase I located on the outer-surface of the mitochondrial inner membrane. Emeriamine also showed hypoglycemic and antiketogenic activities in a dose-dependent manner (1 - 10 mg/kg) when administered orally to fasted normal and diabetic animals.

Clinical implications of telomerase activity in resected hepatocellular carcinoma.

This study investigated the relationship of telomerase activity to its clinical implications in hepatocellular carcinoma (HCC). Telomerase activity was quantatively examined by a non-radioisotope quantitative system based on a TRAP-eze (ONCOR) in 42 HCC nodules and 40 non-cancerous liver tissue adjacent to the HCC nodules, obtained by surgical resection. Telomerase activity was confirmed in 37 HCC nodules (88.1%), being more statistically intense in de-differentiated tumors (p<0.01). Telomerase activity was positive in 9 cases (22.5%) of non-cancerous liver tissue and its activation was closely associated with the biologically malignant potential of the tumor itself such as the histological grade, portal vascular invasion, and intrahepatic metastasis (p<0. 05). The disease-free survival rate with its activity was also statistically lower than that without its activity of the non-cancerous tissue (p<0.01). These results indicate that telomerase may be a useful marker of biological characteristics in HCC and may lead to more effective surgical procedures.

Biological effects and cellular uptake of c-myc antisense oligonucleotides and their cationic liposome complexes.

The biological effects and cellular uptake of human c-myc antisense oligonucleotides and their liposome complexes were investigated in vitro using human promonocytic leukemia U937 cells. Antisense phosphorothioate oligonucleotides (S-Oligo) significantly inhibited the growth of U937 cells in a dose-dependent manner. However, no significant effect on cell proliferation was observed with unmodified phosphodiester (P-Oligo) and partially phosphorothioated (PS3-Oligo) oligonucleotides with an antisense sequence and S-Oligo with sense and G-quartet control sequences. In cellular uptake experiments, radiolabeled S-Oligo was taken up by U937 cells more than P-Oligo and PS3-Oligo. Similar results were obtained in mouse peritoneal macrophages used for comparison. Confocal microscopic studies demonstrated a significant distribution of FITC-labeled oligonucleotides on the cell surface and in the cytoplasm in a punctate pattern, but not in the nucleus. When complexed with cationic liposomes, cellular uptake of FITC-labeled P-Oligo or S-Oligo was significantly increased and the fluorescence was located mainly in the nucleus, indicating that the uptake and intracellular pharmacokinetics of both oligonucleotides can be modified by complexation. An inhibitory effect of the complexes was observed at a dose which is ineffective in the case of the oligonucleotides alone. However, this effect was also associated with cytotoxicity of the cationic liposomes, suggesting that optimization of this formulation will be necessary to achieve a more efficient delivery of the oligonucleotides to U937 cells.

The effect of a nucleotide-nucleoside solution on hepatic regeneration after partial hepatectomy in rats.

After hepatectomy, purine and pyrimidine metabolism is a key process in the synthesis of DNA and RNA and maintaining cellular energy metabolism. The purpose of this study is to evaluate changes in blood purine and pyrimidine levels after partial hepatectomy and the effect of purine and pyrimidine nucleoside solution injection on hepatic regeneration under the hypothesis that the rat after partial hepatectomy requires substrates for salvage nucleotide synthesis and changes blood nucleoside and nucleobase levels. Blood levels of nucleotides, nucleosides, and nucleobase by high-performance liquid chromatography method and liver ATP level by enzymatic analysis, and the effect of preoperative injection of nucleoside solution (OG-VI) on hepatic regeneration ratio and hepatocytes DNA synthesis, were assessed in rats after 70% partial hepatectomy. Decreased liver adenosine triphosphate and increased plasma xanthine and hypoxanthine after partial hepatectomy indicated an increase in catabolism of purine nucleotides in regenerating liver. Plasma thymidine and cytidine levels increased, then returned to the prevalue, suggesting that the thymidine and cytidine pool was enlarged. OG-VI increased labeling indices of hepatocytes at postoperative d 1 (POD) and hepatic regeneration ratio at POD 14. Blood purine nucleobase and pyrimidine nucleoside levels change after partial hepatectomy and preoperative supply of nucleoside solution is effective for increasing hepatocytes DNA synthesis and hepatic regeneration after partial hepatectomy.

Effect of a parenteral nucleoside-nucleotide mixture on hepatic metabolism in partially hepatectomized cirrhotic rats.

After hepatectomy, purine and pyrimidine metabolism is a key process in synthesis of DNA and RNA and in energy metabolism. To supply nucleosides for salvage synthesis, nucleoside-nucleotide mixture solutions have been developed, and they have been found to improve protein metabolism and hepatic regeneration after partial hepatectomy in normal rats. However, the effect of the solution in cirrhotic liver, common in patients with hepatocellular carcinoma, has not been reported. The aim of this study was to evaluate the metabolic effect of the nucleoside-nucleotide mixture on cirrhotic rats after partial hepatectomy. Seventy percent partial hepatectomy was performed in thioacetamide-administered cirrhotic rats. The fractional protein synthetic rate, nitrogen balance, hepatic content of nucleic acid, and blood chemistry after the administration of the nucleoside-nucleotide mixture solution (OG-VI) with total parenteral nutrition was evaluated at 7 d after partial hepatectomy. OG-VI increased hepatic RNA level and hepatic fractional protein synthetic rate (p < 0.05). It is concluded that the nucleoside-nucleotide mixture solution is an effective nutritional supplement to the metabolism of cirrhotic rats after partial hepatectomy.

TAN-931, a novel nonsteroidal aromatase inhibitor produced by Penicillium funiculosum No. 8974. II. Structure elucidation, chemical modification and biological activity.

The structure of TAN-931, a novel nonsteroidal aromatase inhibitor, was determined by chemical reactions and spectral analyses including 2D NMR experiments to be 4-(2,6-dihydroxybenzoyl)-3-formyl-5-hydroxybenzoic acid. Several derivatives of TAN-931 were prepared, and it was found that the 3-formyl and 2'- and/or 6'-hydroxyl groups play an important role in its inhibitory activity. Among the compounds synthesized, 4-(2,6-dihydroxybenzoyl)-3-formyl-5-methoxy-N,N-dimethyl-benzamide was found to be more effective than TAN-931 when administered orally.

Isolation of dealanylalahopcin, a new amino acid, and its biological activity.

A new amino acid, dealanylalahopcin, was isolated from a culture filtrate of Streptomyces albulus subsp. ochragerus; it was also formed by the enzymatic hydrolysis of alahopcin using microbial alpha-amino acid ester hydrolase. The amino acid was obtained as colorless needles and its molecular formula is C6H10N2O5. It showed very weak antibacterial activity against Gram-positive and Gram-negative bacteria, and weak inhibitory activity against the collagen prolylhydroxylase.

Fibrostatins, new inhibitors of prolyl hydroxylase. I. Taxonomy, isolation and characterization.

Fibrostatins, potent inhibitors of prolyl hydroxylase, were isolated as orange crystals from the culture broth of strain No. 23924, which was identified as Streptomyces catenulae subsp. griseospora. In vitro inhibitory activity (ID50 value) of fibrostatins A, B, C, D, E and F against prolyl hydroxylase of chick embryos was 23, 39, 29, 180, 10 and 14 microM, respectively.

Inhibition of prolyl hydroxylase activity and collagen biosynthesis by fibrostatin C, a novel inhibitor produced by Streptomyces catenulae subsp. griseospora No. 23924.

Fibrostatin C, a novel prolyl hydroxylase inhibitor produced by Streptomyces catenulae subsp. griseospora No. 23924, inhibited the activity of purified chick embryo prolyl hydroxylase by about 50% at a concentration of 2.9 x 10(-5) M. The inhibition was mixed type with respect to (Pro-Pro-Gly)5 with a Ki of 2.1 x 10(-5) M. When an excess of ferrous ions or ascorbate was added to the reaction mixture, the inhibition was negligibly or slightly reversed, respectively. Fibrostatin C, when administered intraperitoneally at 1 mg/kg/day or orally at about 100 mg/kg/day as a dietary admixture, significantly inhibited estradiol-17 beta stimulated collagen biosynthesis in the uterus of the immature rat.

TAN-931, a novel nonsteroidal aromatase inhibitor produced by Penicillium funiculosum No. 8974. I. Taxonomy, fermentation, isolation,characterization and biological activities.

A novel nonsteroidal aromatase inhibitor, TAN-931, was isolated from the culture filtrate of a soil isolate fungus, No. 8974. The strain was identified as Penicillium funiculosum No. 8974. TAN-931 inhibited human placental and rat ovarian aromatase activity, and the IC50 value was 17.2 and 162 microM, respectively. The inhibition of human placental aromatase was uncompetitive with respect to androstenedione conversion with a Ki value of 40 microM. When TAN-931 was subcutaneously administered at doses of 25, 50 and 100 mg/kg (once/day, x4) to 20-day-old female Sprague-Dawley rats treated with gonadotropin, the plasma estradiol-17 beta level and the weight of ovaries and uterus were markedly reduced in a dose-dependent manner. The in vivo inhibitory activity of TAN-931 was more potent than that of 4-hydroxyandrostenedione. Consecutive administration of TAN-931 (100 mg/kg, sc, twice/day, x 7) to 9-week-old male Sprague-Dawley rats did not induce any adrenal hypertrophy even though administration of aminoglutethimide caused 2-fold enlargement of the adrenal under the same conditions. Specific binding of TAN-931 to the estrogen receptor from a human breast cancer cell line, MCF-7, was not detected.