Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides.

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.     

A new affinity labeling reagent for the active site of glycogen synthase. Uridine diphosphopyridoxal

A new affinity labeling reagent for glycogen synthase a from rabbit muscle, uridine diphosphopyridoxal, has been prepared. Incubation of the enzyme with this reagent resulted in a time-dependent, almost complete loss of activity. The inactivation was pseudo-first order, and the results of the kinetic analysis suggested the formation of a noncovalent enzyme-reagent complex prior to the covalent reaction, with a Kinact of 25 microM and a maximal rate constant of 0.22 min-1. The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator glucose 6-phosphate. The increase in a spectral peak at 425 nm and the decrease in enzymatic activity were well correlated, suggesting that the reagent causes the inactivation of the enzyme by the formation of a Schiff base. The rate of inactivation increased as the pH was raised, giving a pK of 8.85. Almost all the original activity was recovered by the treatment of the inactivated enzyme with cysteamine or any other aminothiol compound. No recovery of the activity, however, was observed with inactivated enzyme which had been treated with NaBH4. A peptide containing the labeled amino acid was isolated for inactivated enzyme after reduction with NaBH4, carboxymethylation, and chymotryptic digestion by fractionation on a Bio-Gel P-6 column and high performance liquid chromatographies. Manual Edman degradation established the sequence as Glu-Val-Ala-Asn-labeled Lys-Val-Gly-Gly-Ile-(Tyr). The introduction of an active site-directing moiety to pyridoxal 5'-phosphate makes the resultant reagent an effective probe for the active site of glycogen synthase.     

False positive reaction for carcinoembryonic antigen in paget cells

Virchows Archiv B Cell Pathology - Paget cells from cases of mammary and extramammary Paget’s disease were examined for carcinoembryonic antigen (CEA) and CEA-related antigens by the...     

Revised restriction maps of Bacillus subtilis bacteriophage phi 105 DNA.

Physical mapping of Bacillus subtilis temperate phage phi 105 DNA was carried out by using restriction endonucleases EcoRI, SmaI, and KpnI, and a new revised EcoRI cleavage map is presented. In addition, the EcoRI cleavage maps of six specialized transducing phages carrying sporulation genes of B. subtilis were revised.     

Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica.

Mating pheromone-induced alteration of the cell surface proteins of haploid cells, presumed to play crucial roles in the specific cell-cell interactions during sexual conjugation of Tremella mesenterica , was investigated. Exposed surface proteins were revealed by lactoperoxidase-catalyzed iodination in combination with polyacrylamide gel electrophoresis and autoradiography. From comparison of the molecular species of 125I-labeled surface proteins of the vegetative and the gamete (mating pheromone-treated) cells of the two compatible mating types (ab and AB), it was suggested that a striking change in cell surface structure occurs during the differentiation; although labeled protein species of the vegetative cells of the two mating types were indistinguishable, several new species, both mating type specific and nonspecific, appeared in the gamete cells. Turnover of the labeled proteins of the vegetative cells was negligible, whereas that of the gamete cells was rapid with release of low-molecular-weight labeled proteins in the medium. A role for the labeled surface proteins of the gamete cells in the cell-cell interactions during sexual conjugation was suggested by the following: the surface changes were induced by mating pheromone; the labeled proteins were preferentially localized on the surface of the mating tube; the labeled species appeared sequentially during the differentiation; and mating type-specific species were present in both mating types.     

Cytoplasmic effects on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of eight lines of hexaploid wheat (Triticum aestivum L. em. Thell) with different cytoplasms, the euplasmic nuclear donor Chinese Spring and seven alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. The calli were found to differ in their initial growth rates, their sensitivity to 2,4-D and their ability to organise shoot primordia, demonstrating that the cytoplasm can significantly affect the behaviour of tissues in culture. The potential for improving the responses of tissues in culture by cytoplasmic changes is noted.     

Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine