Bioactive oleanolic acid saponins and other constituents from the roots of Viguiera decurrens

The bisdesmoside oleanolic acid saponin, 3-0-(methyl-beta-D-glucuronopyranosiduronoate)-28-0-beta-D-glucopyranosyl-oleanolate along with nine known compounds (two diterpenic acids, one chromene, three triterpenes, one steroidal glycoside, and two monodesmoside oleanolic acid saponins), were obtained from Viguiera decurrens roots. The chemical structure of the bisdesmoside oleanolic saponin was determined by chemical and NMR spectral evidence. A mixture of monodesmoside saponins displayed cytotoxic activity against P388 and COLON cell lines (ED50= 2.3 and 3.6 microg/ml, respectively). Two of the known compounds showed insecticidal activity against the Mexican bean beetle larvae (Epilachna varivestis).     

Effects of ajugarins and related neoclerodane diterpenoids on feeding behaviour of Leptinotarsa decemlineata and Spodoptera exigua larvae

Three naturally occurring ajugarins and seven semisynthetic derivatives of them, possessing different functionalities in the decalin part, together with two natural furoneoclerodane diterpenes, have been assessed as feeding behavior modifying agents of larvae of the generalist Spodoptera exigua and a specialist like Leptinotarsa decemlineata. Ajugarin I and some of its derivatives exhibited a significant antifeedant activity against larvae of S. exigua in both choice and no-choice assays. Conversely, the furoneoclerodane diterpenes only presented antifeedant activity against larvae of L. decemlineata. These results indicate that the biological action of the tested substances is strongly modulated by minimal structural variations, which are also responsible for the specificity of action.     

Analysis of the fatty acid components in a perchloric acid-soluble protein

We had previously found that a perchloric acid-soluble protein (PSP1) occurs in rat liver, and that this novel protein inhibits protein synthesis in a rabbit reticulocyte lysate system (T. Oka, H. Tsuji, C. Noda, K. Sakai, Y.-H. Hong, I. Suzuki, S. Mu?oz, Y. Natori, J. Biol. Chem. 270 (1995) 30060-30067). In the present study, we analyzed lipid components bound to PSP1. Native PSP1 was purified from rat liver using Sephadex G-75, DE-52 cellulose and IgGPSP-affinity chromatography, and the lipid components were extracted. The components obtained from the purified PSP1 were shown to be free fatty acids by thin-layer chromatography. By GC-MS, six major fatty acids were identified as 14:0, 16:0, 18:0, 18:1, 18:2 and 20:4. 1 mol of PSP1 contained 1.26 mol of total fatty acid components. The fatty acid-binding assay of PSP1 showed that the Bmax was 1.25 mol fatty acid/mol PSP1 and the Kd value for palmitic acid was 6.03 microM. The concentration of PSP1 mRNA in rat liver increased 2.3-fold by the administration of peroxisome proliferator, bezafibrate. These findings show that PSP1 is a fatty acid-binding protein-like protein, which is involved in the intracellular metabolism of fatty acid and is quite different from the known fatty acid-binding proteins.     

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity

The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline-regulated expression of beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region-associated, bisected complex oligosaccharides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.     

CCK-mediated response in the activation of 5-HT receptor types in the guinea-pig ileum

The possible interaction between cholecystokinin (CCK) and 5-hydroxytryptamine (5-HT) was evaluated in vitro in the longitudinal muscle-myenteric plexus of the guinea-pig ileum. Devazepide and L-365,260 were used to block CCKA and CCK(B) receptors and ondansetron and tropisetron to block 5-HT3 and 5-HT4 receptors, respectively. The CCK receptor antagonists blocked, in a dose-dependent manner, the response to 5-HT and to the selective agonists at 5-HT3 and 5-HT4 receptors, 2-methyl-5-hydroxytryptamine (2-Me-5-HT) and 5-methoxytryptamine (5-MeOT), respectively. The blockade was almost complete on the first phase of the concentration response curve to 5-HT and for all the concentrations of 5-MeOT tested. In the 2-Me-5-HT-induced contractile response there was a component with the same sensitivity to devazepide and to the selective NK1 receptor antagonist, GR 82334, and another resistant component that was abolished by atropine. However, the blockade of the NK1 receptor did not produce a significant increase in the inhibition obtained when atropine or devazepide were separately tested on the 5-MeOT-induced response. These results suggest that CCK is involved in the 5-HT-induced contractile response, particularly in the response induced by 5-HT4 receptor stimulation.     

Scedosporium apiospermum external otitis.

We report a case of Scedosporium apiospermum external otitis. The patient was topically treated with miconazole cream and achieved a clinical and mycological cure. The etiology, diagnosis and treatment of external fungal otitis are discussed.     

Description of Alaeuris stehlini n. sp. and Alaeuris numidica canariensis n. ssp. (Nematoda, Pharyngodonidae), parasite of Gallotia stehlini, lacertid of Grand Canary Island (Spain)

Pharyngodonid nematodes (Oxyuroidea) belonging to the genus Alaeuris Thapar, 1925, were collected from the posterior gut of Gallotia stehlini (Lacertidae) from Grand Canary Island. Two species Alaeuris stehlini n. sp. and Alaeuris numidica canariensis n. ssp. were identified. The new species is described in which the long thin males are characterized by narrow caudal alae, a rounded first pair of adanal papillae non pedunculate, the second pair attached and elongate, the three pair teated; a short narrow V plate and a relatively long caudal appendage. The females are also long and thin with a slightly salient vulva, a conical pointed caudal appendage, oesophageal length approximately one third of body, excretory pore below the oesophageal bulb. The new subspecies most closely resembles Alaeuris numidica numidica. (Seurat, 1918) Petter, 1966 and Alaeuris numidica madagascariensis Petter, 1966.     

Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.     

Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.     

Cell cycle flow cytometric analysis in the diagnosis and management of colorectal carcinoma

OBJECTIVE: To establish prognostic models and protocols for individualized management in colorectal carcinoma patients based on both clinical and DNA flow cytometric parameters. STUDY DESIGN: Prospective study of 88 colon carcinoma patients with a minimum follow-up of 12 months, operated on with the intent to cure and not treated with radiotherapy or chemotherapy. All the cases were subjected to a clinical evaluation: age, sex, tumor localization and size, histologic grade, tumor stage, disease-free interval, survival and flow cytometric study (ploidy, DNA index and S-phase fraction [SPF]). RESULTS: From the total of 88 neoplasms studied, 56 (63.6%) were from males and 32 (36.4%) from females; 30 (34%) were located in the right side of the colon, 7 (8%) in the transverse colon and 51 (58%) in the left side of the colon. Eleven (12.5%) were stage I, 52 (59.1%) stage II and 25 (28%) stage III. Forty-two (47.7%) were diploid and 46 (52.3%) aneuploid. The S-phase mean was 14.6% (12% for diploids and 16.9% for aneuploids). During the follow-up period, 26.1% of diploid tumors recurred, whereas aneuploid tumors recurred in 36.9% (P < .05). SPF from diploid and aneuploid tumors was analyzed separately. CONCLUSION: Regarding relapse-free interval, the behavior of diploid tumors with a high SPF was similar to that of aneuploid ones. Two kinetic profiles were established, favorable (diploid tumors with low S phase) and unfavorable (diploid with high S phase and all aneuploid tumors), that had significant prognostic value for progression and survival and that allowed identification of patients at high risk of recurrence. We formulated a prognostic index according to SPF and tumor stage that has discriminatory capacity for biologic behavior in colorectal tumors.