Method of identifying inhibitors of oncogenic transformation: selective inhibition of cell growth in serum-free medium.

We developed a new method for evaluating inhibitors of oncogenic signal transduction pathways based on different growth abilities between normal and transformed cells in a defined serum-free medium. The growth rates of src, abl or ras oncogene-transformed cells, activated raf proto-oncogene transformed cells, and normal NIH-3T3 cells were 60-90%, 20-30% and 10% in a serum-free medium, respectively, compared to the growth rates in a serum-containing medium. An addition of a growth factor (PDGF, FGF or TGF-beta) stimulated the growth of normal NIH3T3 cells by 40-80% in a serum-free medium. Herbimycin A, a specific cytoplasmic protein tyrosine kinase inhibitor, selectively inhibited the growth of src or abl transformed cells in the serum-free medium resulting in about 10-fold or fivefold lower IC50 than those in the serum-containing medium. The antibiotic did not show such an effect on ras transformed cells, and the treatment of src transformed cells with other protein kinase inhibitors or cytotoxic drugs showed little IC50 shifts between the two media. Thus, this method of comparing growth inhibition in the serum-free and the serum-containing media may be useful in evaluating specific inhibitors of signaling pathways mediated by growth factors and certain oncogene products.

Log-normal diameter distribution of Pd-based metallic glass droplet and wire.

We have studied the formation of Pd42.5Cu30Ni7.5P20 metallic glass droplets and wires in the gas atomization process. We demonstrate that the sizes of droplets and wires can be distinguished by the Ohnesorge number (Oh), which is the proportion of the spinnability to the capillary instability, and the diameter distributions follow a log-normal distribution function, implying cascade fragmentation. For droplets, the number significantly increases at Oh < 1 but the diameter gradually decreases. For wires, the number greatly increases at Oh > 1 while the diameter steadies below 400 nm. Further, the wire diameter is quadrupled at Oh = 16 due to the high viscosity which suppresses both capillary breakup and ligament elongation.

Further evidence on elevated discrimination limens for reduced patterns in monkeys with inferotemporal lesions.

An attempt was made to elucidate the nature and neural basis of the elevated discrimination limen for visual stimuli of reduced sizes that had been discovered earlier in monkeys with inferotemporal lesions. Although anterior inferotemporal damage was found to have only negligible effect, both total inferotemporal and posterior inferotemporal lesions yielded markedly elevated limens for reduced pattern stimuli but not for reduced colour stimuli. The impaired animals could be trained to discriminate reduced patterns that were below their original discrimination limen, but this required many more trials than were needed for postoperative relearning of the initial pair; normal animals, by contrast, showed perfect transfer from the standard to the smallest pair. The findings suggest that the increased threshold for reduced patterns in monkeys with inferotemporal lesion is attributable not to a visual acuity disorder but to an impairment of pattern perception resulting mainly from damage to posterior inferotemporal cortex.

Sporostatin, a novel and specific inhibitor of EGF receptor kinase.

Sporostatin isolated from a fungus of Sporormiella sp.M5032 as an inhibitor of cyclic adenosine 3',5'-monophosphate phosphodiesterase, was found to be a specific inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase in vitro. Its IC50 values were 0.1 microgram/ml (0.38 microM) for EGF receptor kinase, 3 micrograms/ml (11 microM) for ErbB-2, and 100 micrograms/ml (380 microM) or more than that for other kinases including PDGF receptor, v-src and protein kinase C. Kinetic analyses revealed that inhibition of EGF receptor kinase by sporostatin was noncompetitive either with substrate or with ATP. Autophosphorylation of EGF receptor in A431 cells was also inhibited. These results show that sporostatin is a potent and specific inhibitor of EGF receptor kinase.

Effect of small cue-response separation on pattern discrimination in macaques (Macaca fuscata and M. mulatta).

In order to elucidate the nature of the effect of small cue-response separations on pattern discriminations by monkeys, three studies were performed. When training on a pattern discrimination with a cue-response separation was discontinued during performance at the chance level, there was no saving on the rate of learning a second task (with identical cues but a different cue-response separation) relative to the performance of naive control animals. By contrast, when training was discontinued at a performance level a little better than chance, there was significant saving on learning a second task. After learning the second task, a third task with new pattern cues was learned, with marked saving on the duration of performance at the chance level. The results indicate that during the initial stage of performance at the chance level, monkeys do not attend to cues if there is even a small separation between the cue and the response site.

Acquisition of discrimination learning of patterns identical in configuration in macaques (Macaca mulatta and M. fuscata).

The acquisition of discrimination of five pairs of pattern cues in a Wisconsin General Testing Apparatus by 223 naive macaque monkeys was compared. The pairs of discriminanda were identical in configuration but varied slightly in either the size of the cue or the size of the background plaque; thus, the degree of separation of the cue from the fringe of the plaque, the response site, was slightly different for each pair of discriminanda. These small differences in cue-response separation had marked effects on the rate of acquisition of the discriminations. Even an increment of separation as small as 0.5 cm resulted in a remarkable retardation of the acquisition. This retardation was due entirely to prolonged performance at the chance level, and not to a slow rate of improvement from the chance to a criterion level. The finding indicates that the difficulty in the acquisition learning on pattern tasks depends largely on the difficulty of attending to the pattern cues at small cue-response separations.

Studies on neplanocin A, new antitumor antibiotic. II. Structure determination.

The structure and stereochemistry of neplanocin A was determined on the basis of its spectral and chemical evidences as [1R-(la,2a,3b)]-3-(6-amino-9-yl)-5-(hydroxy-methyl)-4-cyclopentene-1,2-diol. For the final proof of the structure, X-ray crystallographic analysis of neplanocin A was carried out.

Studies on neplanocin A, new antitumor antibiotic. I. Producing organism, isolation and characterization.

Neplanocin A. C11H13N5O3, is a novel carbocyclic analog of adenosine with cyclopentene. It was isolated from the culture filtrate of Ampullariella regularis A11079 by means of ion-exchange, carbon, silica gel adsorption, or partition chromatography. Neplanocin A forms crystals, and is stable at acidic or alkaline pH. Neplanocin A has cytotoxicity against L5178Y cells in culture and showed a remarkable effect on the life prolongation of mice infected with L1210 leukemia.

Isolation and characterization of new thiol protease inhibitors estatins A and B.

New thiol protease inhibitors, estatins A and B, were isolated from the culture filtrate of Myceliophthora thermophila M4323. The basic, water-soluble inhibitors were characterized as having an agmatine, trans-epoxysuccinic acid and L-phenylalanine or L-tyrosine moieties in the structure. The molecular formulas C18H25N5O5 and C18H25N5O6 for A and B were indicated by elemental analysis and fast atom bombardment MS. Estatins were specific inhibitors against thiol proteases such as papain, ficin and bromelain. They suppressed IgE antibody production in mice, but not IgG.

Sporeamicin A, a new macrolide antibiotic. I. Taxonomy, fermentation, isolation and characterization.

Sporeamicin A, a novel antibiotic, was isolated from the culture filtrate of an actinomycete. The producing organism, strain L53-18, was taxonomically assigned as a species of the genus Saccaropolyspora. The antibiotic was extracted with chloroform and was then purified by crystallization. It was obtained as colorless prisms from ethanolic solutions. Sporeamicin A exhibited a strong UV absorption peak at 276 nm. The molecular formula of sporeamicin A was determined to be C37H63NO12.

Sporeamicin A, a new macrolide antibiotic. II. Structure determination.

Structure of a novel antibiotic, sporeamicin A (SRM-A), was determined by a combination of spectroscopic and X-ray crystallographic studies. SRM-A has a unique structure containing a 2,3-dihydro-3-oxofuran moiety as part of a 14-membered macrolide ring.

Sporeamicin A, a new macrolide antibiotic. III. Biological properties.

Sporeamicin A is a new erythromycin-type antibiotic isolated from a species of Saccharopolyspora. It was active in vitro against a wide variety of Gram-positive bacteria. In vitro studies indicated that the sporeamicin A was stable in the presence of human serum, although it was bound to serum proteins. Sporeamicin A was effective in the mouse protection test against Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae. Sporeamicin A attained higher plasma and tissue levels in the rat than did erythromycin stearate.

Stachybocins, novel endothelin receptor antagonists, produced by Stachybotrys sp. M6222. II. Structure determination of stachybocins A, B and C.

The structures of stachybocins A, B and C, new endothelin receptor antagonist, were determined by NMR spectral analysis using pulse-field-gradient technique. Stachybocin A consists of two spirobenzofuran units each fused to a substituted decalin, which were connected by a lysine residue. Stachybocins B and C are derivatives of stachybocin A with an additional hydroxy group at the same position in the different decalin unit.

Stachybocins, novel endothelin receptor antagonists, produced by Stachybotrys sp. M6222. I. Taxonomy, fermentation, isolation and characterization.

Stachybocins A, B and C, novel endothelin (ET) receptor antagonists, were isolated from the culture filtrate of Stachybotrys sp. M6222. They were extracted with ethyl acetate and then purified by alumina and silica gel column chromatographies. The molecular formulae of stachybocins were determined to be C52H70N2O10 (stachybocin A) and C52H70N2O11 (stachybocins B and C). It was supposed that they consisted of spirobenzofuran and terpene units from NMR spectra. They showed the inhibitory activity of 125I-ET-1 binding to rate ETA, human ETA and human ETB receptors.