Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcγRs.

Enhancing the effector function by optimizing the interaction between Fc and Fcγ receptor (FcγR) is a promising approach to enhance the potency of anticancer monoclonal antibodies (mAbs). To date, a variety of Fc engineering approaches to modulate the interaction have been reported, such as afucosylation in the heavy chain Fc region or symmetrically introducing amino acid substitutions into the region, and there is still room to improve FcγR binding and thermal stability of the CH2 domain with these approaches. Recently, we have reported that asymmetric Fc engineering, which introduces different substitutions into each Fc region of heavy chain, can further improve the FcγR binding while maintaining the thermal stability of the CH2 domain by fine-tuning the asymmetric interface between the Fc domain and FcγR. However, the structural mechanism by which the asymmetrically engineered Fc improved FcγR binding remained unclear. In order to elucidate the mechanism, we solved the crystal structure of a novel asymmetrically engineered Fc, asym-mAb23, in complex with FcγRIIIa. Asym-mAb23 has enhanced binding affinity for both FcγRIIIa and FcγRIIa at the highest level of previously reported Fc variants. The structural analysis reveals the features of the asymmetrically engineered Fc in comparison with symmetric Fc and how each asymmetrically introduced substitution contributes to the improved interaction between asym-mAb23 and FcγRIIIa. This crystal structure could be utilized to enable us to design a more potent asymmetric Fc.

Effective interaction energy of water dimer at room temperature: an experimental and theoretical study.

Buffer-gas pressure broadening for the nu(1)+nu(3) band of H(2)O at 1.34-1.44 mum for a variety of buffer gases was investigated at room temperature using continuous-wave cavity ring-down spectroscopy. The effective interaction energy of water dimer under room temperature conditions was evaluated from the pressure broadening coefficients for rare gases using Permenter-Seaver's relation. Monte Carlo simulations were performed using ab initio molecular orbital calculations to evaluate the interaction energies for the water dimer at 300 K. In this theoretical calculation, the orientations of the two water molecules were statistically treated.

Comprehensive allelotyping of human intrahepatic cholangiocarcinoma.

We performed a genome-wide scan for loss of heterozygosity (LOH) in 22 intrahepatic cholangiocarcinoma (ICC) cases using 168 polymorphic microsatellite markers throughout all of the human chromosomes and 48 markers of which LOH is reportedly characteristic of hepatocellular carcinoma (HCC). Markers with LOH in more than 30% of informative cases were observed at 21 loci. Among these, eight markers on 6q (three loci), 4q (two loci), 9q, 16q, and 17p shared high frequencies of LOH with HCC in our previous study. As for gross appearance, mass-forming type tumors showed higher frequency of LOH (P < 0.001) compared with other types. Compared by tumor size (< or =5 cm versus >5 cm), number (multiple versus solitary), and the International Union Against Cancer TNM classification (stage IVB versus II-IVA), LOH was observed more frequently in advanced stages (P < 0.01, respectively). However, LOH frequency does not differ regardless of lymph node status (pN0 versus pN1). Frequent LOH on 1p36 including the p73 locus was noted in large tumors without lymph node metastasis. These suggest that ICC shares some common carcinogenic steps with HCC such as LOH of 4q and 6q and that inactivation of tumor suppressor genes on chromosome 1p36 contributes to progression of ICC but not to metastatic traits.

Increased expression of cytosolic chaperonin CCT in human hepatocellular and colonic carcinoma.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTbeta in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTbeta antibody. The levels of CCTbeta were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTalpha gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.

Comprehensive allelotype study of hepatocellular carcinoma: potential differences in pathways to hepatocellular carcinoma between hepatitis B virus-positive and -negative tumors.

To examine the role of the loss of heterozygosity (LOH) in hepatitis-related carcinogenesis, we performed a genome-wide scan of LOH in 44 tumors of hepatocellular carcinoma (HCC) using 216 microsatellite markers throughout all human chromosomes. A high frequency of LOH (>30% of informative cases) was observed at 33 loci on chromosome arms 4q, 6q, 8p, 8q, 9p, 9q, 13q, 16p, 16q, 17p, and 19p. LOH on 19p has not yet been reported, and that appears to be a new candidate in the search for tumor suppressor genes. High rates of LOH are correlated with hepatitis B virus (HBV) positivity, poorly differentiated tumors, vascular invasion, and intrahepatic metastasis (P <.0001). LOH on 13q and 16q occurred more frequently in HBV(+) patients (P <.0001), and LOH on 6q occurred more frequently in virus-negative patients (P <.001). The frequency of LOH on 4q and 13q was significantly lower in well-differentiated tumors than in moderately and poorly differentiated tumors (P <.01). In contrast, LOH on 6q was frequently detected in well-differentiated tumors compared with other histological subclasses (P <.001). Our results suggest that LOH on 6q may play an important role in the early stage of hepatocarcinogenesis in virus-negative patients, but different mechanisms might underlie the initial step to carcinogenesis in HBV(+) patients. LOH on 13q and 16q may play an essential role in the progression of HBV(+) tumors. Further studies of fine deletion mapping on chromosomes 13q and 16q are required to define the genomic segments on which putative tumor suppressor genes responsible for HBV(+) tumors exist.

Molecular design of antibrowning agents.

Tyrosinase inhibitory and antioxidant activity of gallic acid and its series of alkyl chain esters were investigated. All inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase. However, gallic acid and its short alkyl chain esters were oxidized as substrates yielding the colored oxidation products. In contrast, the long alkyl chain esters inhibited the enzyme activity without being oxidized. This indicates that the carbon chain length is associated with their tyrosinase inhibitory activity, presumably by interacting with the hydrophobic protein pocket in the enzyme. On the other hand, the esters, regardless their carbon chain length, showed potent scavenging activity on the autoxidation of linoleic acid and 1,1-diphenyl-2-p-picryhydrazyl (DPPH) radical, suggesting that the alkyl chain length is not related to the activity. The effects of side-chain length of gallates in relation to their antibrowning activity are studied.

Analysis of genetic changes in intrahepatic cholangiocarcinoma induced by thorotrast.

Thorotrast, a colloidal suspension of radioactive (232)ThO(2) that emits alpha particles, was used as a radiographic contrast agent in the 1930s-1950s. Several decades after injection, Thorotrast causes liver cancers, among which intrahepatic cholangiocarcinoma (ICC) is prominent. We investigated mutations of the RAS and the TP53 genes in archival sections of ICC induced by Thorotrast. Compared to ICC that was not associated with Thorotrast, the frequency of mutation of the KRAS gene was lower, while that of the TP53 gene was more than two times higher. The most common mutation of the TP53 gene was A-G transitions. Interestingly, TP53 mutations were also found in noncancerous areas of livers in which Thorotrast had been deposited. Furthermore, mutations tended to accumulate in tissues from more advanced tumors. These results suggest that deposited Thorotrast continuously damages DNA in liver cells in some way, resulting in A-G transitions of the TP53 gene. However, we have not been able to rule out the possibility that genetic insults occur indirectly in the proliferating cells adjacent to the necrosis rather than being a direct effect of alpha particles.

Expression of the thymidine phosphorylase gene in epithelial ovarian cancer.

Thymidine phosphorylase (TP) is associated with angiogenesis and the progression of solid tumours. High intracellular levels of this enzyme indicate increased chemosensitivity to pyrimidine antimetabolites. TP gene expression in 56 cases of epithelial ovarian cancer (27 of serous, 10 mucinous, 12 endometrioid, five clear cell and two undifferentiated) were analysed by polymerase chain reaction of RNA after reverse transcription. These included eight of low malignant potential. Twenty were stage I, four stage II, 27 stage III and five stage IV. The level of TP gene expression was presented by the relative yield of the TP gene to the beta2-microglobulin gene. TP gene expression ranged from 0.19 to 5.38 (median 0.93). The value of TP gene expression in stage III-IV was significantly higher than that of TP gene expression in stage I-II (P = 0.0005). Histological grade significantly associated with TP gene expression (P = 0.008), but histological subtype did not (P = 0.166). A follow-up study of 34 cases after complete resection of the primary tumours by surgical operation was performed. TP gene expression of the cases with recurrence showed significantly higher levels compared to cases without recurrence (P = 0.049). Survival data were available for 47 of the 56 patients. The prognosis of the patients with high TP gene expression (equal to, or greater than, median) was to be significantly worse than patients with low TP gene expression (less than median) (P = 0.021). The TP gene expression level may play one of the key roles in the biology of ovarian epithelial cancer and define a more aggressive tumour phenotype. A new therapeutic intervention mediated by TP protein activity is anticipated.

Sulfated glycoglycerolipid from archaebacterium inhibits eukaryotic DNA polymerase alpha, beta and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity.

A sulfated glycoglycerolipid, 1-O-(6'-sulfo-alpha-D-glucopyranosyl)-2,3-di-O-phytanyl- sn-glycerol (KN-208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) alpha and pol beta in vitro among 5 eukaryotic DNA polymerases (alpha, beta, gamma, delta, and epsilon). It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E. coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT). The mode of inhibition of these polymerases was competitive with the DNA template primer and was non-competitive with the substrate dTTP. KN-208 inhibited pol beta most strongly, with a Ki value of 0.05 microM, 10-fold lower than that for pol alpha (0.5 microM) and 60- or 140-fold lower than that for HIV RT (3 microM) or for E. coli pol I (7 microM), respectively. The loss of sulfate on the 6'-position of glucopyranoside of this compound completely abrogated inhibition. However, the hydrophilic part of KN-208, glucose 6-sulfate alone, showed no inhibition. Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition. Our results suggest that the whole molecular structure of KN-208 is required for inhibition. KN-208 was shown to be modestly cytotoxic for the human leukemic cell line K562. Interestingly, a subcytotoxic dose of KN-208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin.

SRS-A antagonist pyranoquinolone alkaloids from east African Fagara plants and their synthesis.

Three pyranoquinolone alkaloids isolated from two East African Fagara plants have been found to exhibit SRS-A antagonist action. Their synthesis has been accomplished, using a modified Coppola's method or a thermal cyclization followed by an electrocyclic ring closure.

Synthesis of 2,3-di-O-phytanyl-1-O-(alpha-D-glucopyranosyl)-sn-glycerol derivatives, analogues of polar lipids isolated from a halophilic bacterial strain.

2,3-Di-O-phytanyl-1-O-glucopyranosylglycerol and polar derivatives of its 6'-glucose moiety have been synthesized. The target molecule contains the diphytanyl-sn-glycerol moiety which is alpha-linked to glucose. The key step in its synthesis involves the coupling of phytanyl bromide and isopropylidene threitol. We also demonstrated that the 6'-hydroxyl group of glycolipids can be functionalized without protection of the sugar moiety.

Significance of pre-existent conditions in basal cell carcinoma on the lower extremities.

BACKGROUND: Basal cell carcinoma (BCC) rarely appears on the lower extremities; over 85% appear on the head and neck. CASE REPORTS: Five patients had BCC on the lower extremity. Histologically, this was of the superficial type. One patient developed a lesion of actinic keratosis. RESULTS: The presence of pre-existent cutaneous changes of BCC arising on the lower extremities was studied. Among 40 tumors reported in Japan, 10 cases (25%) had pre-existent cutaneous changes. This value is significantly higher than those of BCC over the whole body in both Japanese and Caucasian. CONCLUSIONS: Pre-existent cutaneous changes, thus, are thought to be a possible etiologic factor in the pathogenesis of BCC on the lower extremities.

Molluscicidal acridone alkaloids from Angostura paniculata: isolation, structures, and synthesis.

Two novel acridone alkaloids, cuspanine [1] and cusculine [2], were isolated from the CH2Cl2 extract of the leaves of Angostura paniculata (Rutaceae). Their structures were established as 1-hydroxy-2,3,5,6-tetramethoxy-9-acridone for 1 and 1,2,3,5,6-pentamethoxy-9-acridone for 2 by means of spectroscopic studies, in particular nmr. These structural assignments were confirmed by synthesis, using a direct metallation method as a key reaction. Both alkaloids exhibited moderate molluscicidal activity against an aquatic snail, Biomphalaria glabrata, and cytotoxicity against several types of carcinoma cell lines.

Solitary trichoepithelioma arising in the elbow.

A 52-year-old Japanese woman with solitary trichoepithelioma on the left elbow was described. This location of the tumor is exceptional.

Isolation, structure, and synthesis of novel 4-quinolinone alkaloids from Esenbeckia leiocarpa.

Two new biocidal quinolinone alkaloids, 3-methoxy-1-methyl-2-propyl-4-quinolone [1] and 2(1'-ethylpropyl)-1-methyl-4-quinolone [2], were efficiently isolated using reversed-phase recycling hplc from the leaves of Esenbeckia leiocarpa. The structures were determined through spectroscopic data and confirmed by total synthesis. These alkaloids have antifeedant activities against the pink bollworm, Pectinophora gossypiella.

Carnitine derivatives in hereditary cardiomyopathic animals.

BIO 14.6 Syrian hamsters and diabetic KK mice have been reported to develop hereditary cardiomyopathy spontaneously. In order to investigate the pathophysiological role of carnitine metabolism in hereditary cardiomyopathy, tissue levels of carnitine derivatives and the histology of the heart, liver and skeletal muscles from BIO 14.6 hamsters and KK mice were studied. Free carnitine levels in the heart of the BIO 14.6 hamsters (287.0 +/- 27.0 n mole/g wet tissue) were significantly lower than in the control group (348.8 +/- 83.8, p less than 0.05). Short chain acylcarnitine (197.0 +/- 56.0 n mole/g wet tissue) and total carnitine (667.6 +/- 136.4 n mole/g wet tissue) in the hearts of the BIO 14.6 hamsters were significantly lower than in the control group (short: 425.2 +/- 54.8, total: 1023.6 +/- 81.4, p less than 0.001). There was no significant difference in the levels of various carnitine derivatives of the liver and skeletal muscles from the BIO 14.6 hamsters and control hamsters. On the other hand, carnitine derivatives in KK mice did not change significantly compared with those in the heart, liver and skeletal muscles of the control mice. Histological findings showed that heart muscle degeneration and necrosis were found in both cardiomyopathic animals. Coagulative necrosis was found in both animals, whereas myocytolytic necrosis was found only in the BIO 14.6 hamsters. In KK mice, the right ventricle, especially tissue under the epicardium, was severely affected compared with the left ventricle. In the BIO 14.6 hamsters, however, lesions were scattered over both ventricles with a predilection for the left ventricle.

Effects of coenzyme Q10 on exercise tolerance in chronic stable angina pectoris.

The effects of coenzyme Q10(CoQ10) on exercise performance were studied in 12 patients, average age 56 years, with stable angina pectoris. The study involved a double-blind, placebo-controlled, randomized, crossover protocol, using multistage treadmill exercise tests. CoQ10(150 mg/day in 3 daily doses) was administered orally for 4 weeks, tended to reduce anginal frequency from 5.3 +/- 4.9 to 2.5 +/- 3.3 attacks for 2 weeks and nitroglycerin consumption from 2.6 +/- 2.8 to 1.3 +/- 1.7 tablets for 2 weeks compared with patients receiving the placebo, but the reduction was not statistically significant. Exercise time increased from 345 +/- 102 seconds with placebo to 406 +/- 114 seconds during CoQ10 treatment (p less than 0.05). The time until 1 mm of ST-segment depression occurred increased from 196 +/- 76 seconds with placebo to 284 +/- 104 seconds during CoQ10 treatment (p less than 0.01). During the exercise test, ST-segment depression, heart rate and pressure-rate product at the same and at the maximal workload showed no significant difference between patients after placebo and CoQ10 administration. The average CoQ10 plasma concentration increased from 0.95 +/- 0.48 microgram/ml to 2.20 +/- 0.98 microgram/ml after CoQ10 treatment. This increase was significantly related to the increase in exercise duration (r = 0.68, p less than 0.001). Only 1 patient had a loss of appetite, but continued therapy. This study suggests that CoQ10 is a safe and promising treatment for angina pectoris.

Effect of nitroglycerin on left ventricular function in patients with ischemic heart disease.

To evaluate the effects of nitroglycerin on left ventricular function in 27 patients with ischemic heart disease, ejection fraction (EF) was measured every 1 to 5 min by a nuclear stethoscope after sublingual administration of nitroglycerin (0.3 mg). There was a good correlation between EF determined by the nuclear stethoscope and EF by left ventriculography (r = 0.80, p less than 0.001). EF showed a rise after sublingual nitroglycerin, which was most marked at 4 to 7 min and returned to the control level in 20 to 25 min. There were no significant differences in the maximum percent increase in EF among patients with 0, 1, 2 and 3 vessel disease. The maximum percent increases in EF were 34.0 +/- 10.0% in the normal contraction group, 24.0 +/- 8.5% in the hypokinesis group (p less than 0.05 vs the normal contraction group) and 15.2 +/- 8.5% in the akinesis group (p less than 0.01 vs the normal contraction group, p less than 0.05 vs the hypokinesis group). There was a weak correlation between the maximum percent increase in EF and the changes in heart rate (r = 0.49, p less than 0.05) and there was an inverse correlation between the maximum percent increase in EF and the changes in systolic blood pressure (r = -0.65, p less than 0.01). It was shown that the improvement in EF by sublingual nitroglycerin was greatest in the normal contraction group, somewhat less in the hypokinesis group and least in the akinesis group. The nuclear stethoscope is useful in monitoring changes in left ventricular function during intervention.