New taxol assay method using tubulin-assembly stimulation

Summary A novel taxol determination method which involves the tubulin-assembly stimulation is described. The tubulin-assembly was monitored by turbidity change at 350nm. In a limited range of taxol concentration (0 to 24 M), taxol stimulated tubulin-assembly linearly. And this linear relation was observed from 20min to 30min after the reaction started. Bioactive derivatives of taxol, such as cephalomanin and 7-epi-10-deacetyltaxol also stimulated the tubulin-assembly. However, baccatin III, which was known as less active taxol derivative did not stimulate tubulin assembly. This result showed that the stimulation of tubulin assembly has a relationship with the antimiotic activity. This assay method have several advantages. 1) Time required for the measurement is relatively short. 2) Multiple samples can be measured simultaneously. 3) It can remove interference of less active taxane compounds more selectively than immuno-assay. Consequently, this method can be used to determine taxol concentration in biological samples. Especially, this method can be used for large scale selection of cell line and primary screening of new antimiotic compounds.     

Location of the active site of the bean {alpha}-amylase inhibitor and involvement of a Trp, Arg, Tyr triad

Seeds of the common bean contain three homologous proteins:phytohaemagglutinin E, phytohaemagglutinin L and the lectin-likeprotein     

The physiology of the ovary: Maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3–7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate β-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with β-carotene reduced the amount of cross-linking. We conclude that the high levels of β-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.     

Proton nuclear magnetic resonance spectroscopy of isolated rabbit fundic glands

1H-nuclear magnetic resonance spectroscopy (NMR) was adapted to isolated rabbit fundic glands and identification made of compounds responsible for several observed spectral resonances. A minimum gland concentration of 0.5 mg dry weight or 5 mg wet weight per 0.5 ml was needed for adequate signal-to-noise ratio. At physiological temperature and pH, the glands demonstrated reproducible spectra, stability for accumulation times greater than 30 min and responsiveness to histamine stimulation, as measured by oxygen consumption and aminopyrine uptake. The relatively anaerobic conditions favored use of proton compared to phosphorus NMR, since 1H-NMR allowed significantly shorter spectral accumulation times and therefore did not compromise glandular viability to the same extent as 31P-NMR. The most conspicuous resonance in the gland spectrum was assigned to the -N+(CH3)3 protons of choline and related compounds. In membrane-free lysates, several components of the signal were resolvable and assigned to choline, phosphatidylcholine, phosphocholine and L-alpha-glycerophosphocholine. Thin-layer chromatography verified that phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present in gland lipid. Presumably, they represent the source of the surface-active phospholipids present in gastric juice, which may play a role in gastric cytoprotection.     

Infrared spectra of carbon monoxide bound to mitochondria from diverse species and tissues reveal structurally similar cytochrome c oxidase dioxygen reaction sites

Infrared bands for CO bound to mitochondria from bovine and porcine hearts, bovine brain, rat kidney, and blowfly flight muscle and to intact blowfly flight muscle have been measured in the carbon-oxygen stretch region. Each spectrum contains a narrow band near 1963 cm-1 similar to the major band found earlier for the carbonyl cytochrome c oxidase purified from bovine heart. A second band near 1959 cm-1 ascribed to a less stable conformer of the purified oxidase carbonyl is also detected in mitochondria. These spectra support very similar CO (and O2) binding sites among all the oxidases examined whether the enzyme is purified or is still within mitochondria or intact tissue and therefore suggest that the reduced heme A ligand binding site has been highly conserved during evolution.     

Ferritin conjugates as specific magnetic labels. Implications for cell separation

Concanavalin A coupled to the naturally occurring iron storage protein ferritin is used to label rat erythrocytes and increase the cells' magnetic susceptibility. Labeled cells are introduced into a chamber containing spherical iron particles and the chamber is placed in a uniform 5.2 kG (gauss) magnetic field. The trajectory of cells in the inhomogeneous magnetic field around the iron particles and the polar distributions of cells bound to the iron particles compare well with the theoretical predictions for high gradient magnetic systems. On the basis of these findings we suggest that ferritin conjugated ligands can be used for selective magnetic separation of labeled cells.     

Needle aspiration cytology of a benign and a malignant schwannoma

The histologic and cytologic features of an uncommon solitary, malignant schwannoma and a benign schwannoma are presented. The benign tumor revealed characteristic interlacing fascicles of spindle-shaped cells on both histology and cytology. The malignant schwannoma produced a variable histologic pattern that was selectively sampled from a recurrence in a scar; only obviously malignant, undifferentiated, spindle-shaped cells not capable of further characterization were seen on cytology. Preliminary experience with immunoperoxidase staining for the neural crest marker S-100 protein has been encouraging and may permit identification of these tumors on aspiration smears.     

On the evolution of cicada X host-tree associations in Central America

An hypothesis tout court is proposed to account for the association of cicadas (Homoptera: Cicadidae) with large-sized trees of the family Leguminosae in both climax forest formations and forest remnants in Central America. Based upon extensive field studies of these insects in Costa Rica, final-instar nymphal skins of many genera and species of Neotropical cicadas appear to be aggregated annually around individual large legume trees in a broad range of habitats in Costa Rica, and much of the reproductive behavior of the adult insects is also centered around these trees. A series of testable subhypotheses are generated based largely upon the assumption that cicadas, primarily through egg-placement behavior and nymphal feeding behavior, have coevolved with legume trees in climax forest formations of Central America, and perhaps elsewhere in the Neotropical Region. The root crowns of such trees, with some exceptions, might be optimal feeding sites for cicada nymphs in tropical forests.     

Reduced maximum capacity of glycolysis in brown adipose tissue of genetically obese, diabetic (db/db) mice and its restoration following treatment with a thermogenic beta-adrenoceptor agonist

The maximal activities of the key glycolytic enzymes hexokinase and 6-phosphofructokinase, were reduced in brown adipose tissue in db/db mice compared to their lean littermates. Treatment of db/db mice with the thermogenic beta-adrenoceptor agonist, BRL 26830, restored normoglycaemia. The only significant increase in activity of hexokinase and 6-phosphofructokinase in the BRL 26830-treated db/db mice occurred in brown adipose tissue where the total tissue activity increased 10- and 11-fold respectively. These changes together with increased 2-deoxyglucose uptake in vivo suggest that brown adipose tissue can play a quantitatively important role in the removal of glucose from the blood.     

Identification of Mr variants of proclatin with monoclonal antibodies

Monoclonal antibodies (QB01 and 1200) prepared against human proclatin (hPRL) have helped define a variant form of the hormone. This variant is of apparently higher molecular mass (26kDa) than the predominant form of the hormone (24kDa) and its presence does not appear to be species-restricted. The demonstration of the 26 kDa form of the hPRL in fresh pituitary tissue and amniotic fluid suggests it may retain some specific function.