Alkaloid production in cultured cells of Dioscoreophyllum cumminsii

Jatrorrhizine and magnoflorine were isolated from cultured cells of Dioscoreophyllum cumminsii. The jatrorrhizine content in cultured cells was 40–100 times higher than that of the intact plant, but columbamine, which is a minor component in the original plant, was not detected. Moreover, it was observed that the addition of 1AA or NAA to the growth medium increases the alkaloid content as compared with 2,4-D.     

Stimulation of chalcone synthase activity by yeast extract in cultured Glycyrrhiza echinata cells and 5-deoxyflavanone formation by isolated protoplasts

Chalcone synthase activity catalyzing the formation of naringenin (5-hydroxyflavanone) was detected in cell suspension cultures of Glycyrrhiza echinata. This activity rapidly increased by treatment of the cells with yeast extract, while non-treated cells showed a constant low activity. Isolated G. echinata protoplasts accumulated retrochalcone (echinatin) and its biosynthetic intermediate (licodione) during 24 h of culture. When the protoplasts were incubated with [¹⁴C(U)]phenylalanine, liquiritigenin (5-deoxyflavanone) was transiently labeled, indicating the induction of 6'-deoxychalcone synthase. The formation of liquiritigenin, in addition to naringenin, was observed when the crude extracts from the protoplasts were assaved for CHS activity.Abbreviations CHS chalcone synthase - YE yeast extract This paper is Part 52 in the series Studies on Plant Tissue Cultures. For Part 51, see Furuya T, Ushiyama M, Asada Y, Yoshikawa T, Orihara Y (1987) Phytochemistry: in press.     

Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium

Summary Growth hormone (GH) production by GH₁ rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T₃). As measured by radioimmunoassay, apoTf plus T₃ induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T₃ arrested GH secretion. ApoTf/T₃ defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T₃ defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T₃ defined medium. Even under these conditions, the response to dexamethasone remained T₃ dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the reponse to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.     

Mechanically induced electrical and intracellular calcium responses in normal and cancerous mammary cells.

Mechanically induced channel activities and increase of intracellular calcium ([Ca2+]i) in normal and cancerous murine mammary cells (MMT 060562) were investigated using the patch clamp technique and Fura-2 fluorescence. Both cell types showed similar properties. Upon mechanical stimulation, activation of the Ca(2+)-dependent K+ channel or outward membrane current was recorded in cells which were several cells distant from the stimulated cell. Mechanical stimulation also induced an increase of [Ca2+]i in the touched cell, and this increase of [Ca2+]i spread to the surrounding cells. The [Ca2+]i signal travelled a distance of 100-200 microns within 20-40 s and then diminished. The presence of cell-to-cell communication between adjacent mammary cells through gap junction was indicated by injection of lucifer yellow and measurements of electrical coupling (coupling constant = 0.2-0.3). The mechanically induced increase of the [Ca2+]i signal spread to adjacent cells even when the stimulated cell had no physical contact with them. In the absence of fluid movement, the pattern of the spread of the [Ca2+]i signal was a concentric circle. However, in the presence of fluid movement, the pattern changed to elongate to the direction of the flow. These findings suggested that a certain factor was released from the mechanically stimulated cell to the extracellular space, and this factor induces the increase of [Ca2+]i in surrounding cells.     

Identification of GTP-Binding Proteins by ADP-Ribosylation in the Presence of Cholera Toxin, Pertussis Toxin and Botulinum Toxin D in Plasma Membrane Isolated from Eggs and Embryos of Sea Urchin

In plasma membrane fraction isolated from eggs and embryos of sea urchin, ³²P-labeled proteins were found on the fluorographs of SDS-polyacrylamide gel electrophoresis, performed after an exposure of the fraction to [adenylate-³²P] nicotinamide adenine dinucleotide in the presence of cholera toxin, pertussis toxin or botulinum toxin D. The molecular weights of proteins, thus ADP-ribosylated in the presence of cholera toxin and pertussis toxin are 45 and 39 K, which correspond to Gs and Gi or Go, respectively. Protein with the molecular weight of 24 K, labeled in the presence of botulinum toxin D, corresponds to small molecular weight G-protein. The labeling intensity of 45 K protein, probably proportional to its amount, became high at the blastula stage. The labeling intensity of 39 K protein was hardly altered up to the blastula stage. The labeling intensity of 24 K protein increased after fertilization and further increase occurred at the blastula stage. At the gastrula stage, the labeling intensities of these proteins became somewhat lower than at the blastula stage. Transmembrane signaling system, in which these G-proteins are involved, is probably altered in its function during early development.     

Differential usage of photoreceptors for chalcone synthase gene expression during plant development

Photoregulation of chalcone synthase (CHS) mRNA accumulation was analysed in parsley (Petroselinum crispum) and mustard (Sinapis alba) plants at different developmental stages. In both species, CHS mRNA accumulation in young etiolated seedlings was primarily under phytochrome control. In leaves of adult re-etiolated plants, a UV-B photoreceptor was predominantly involved in photocontrol. The reduced red light control in mature leaves was not due to the absence of immunoreactive phytochrome. The apparent dependence of photoreceptor usage on the developmental state of the cell or organism was in accordance with observations on the photoregulation of fusion constructs between CHS promoters from parsley or mustard and the β-glucuronidase reporter gene (GUS). When tested in the parsley protoplast transient expression system, both constructs yielded the same type of photoregulation as observed for the endogenous CHS gene.     

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.     

Identification of feline immunodeficiency virus rev gene activity.

We constructed 16 deletion mutants from an infectious molecular clone of feline immunodeficiency virus (FIV) and a reporter plasmid carrying the bacterial chloramphenicol acetyltransferase (CAT) gene to identify the rev transactivator activity of the virus. Cotransfections of various mutants and the rev reporter clone bearing a portion of FIV env in addition to the CAT gene revealed that the sequence important for the augmentation of CAT production was located in three separate parts of the virus genome. This enhancement was FIV specific in that the human retrovirus rev and rex gene products did not activate the reporter. The phenotypic properties of an FIV proviral mutant containing a small deletion in the genome were similar to those of rev mutants derived from primate immunodeficiency viruses. These results indicate that FIV, like the other lentiviruses, contains the rev gene in its genome.