Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.     

Elementary Functional Properties of Single HCN2 Channels

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are tetramers that evoke rhythmic electrical activity in specialized neurons and cardiac cells. These channels are activated by hyperpolarizing voltage, and the second messenger cAMP can further enhance the activation. Despite the physiological importance of HCN channels, their elementary functional properties are still unclear. In this study, we expressed homotetrameric HCN2 channels in Xenopus oocytes and performed single-channel experiments in patches containing either one or multiple channels. We show that the single-channel conductance is as low as 1.67 pS and that channel activation is a one-step process. We also observed that the time between the hyperpolarizing stimulus and the first channel opening, the first latency, determines the activation process alone. Notably, at maximum hyperpolarization, saturating cAMP drives the channel to open for unusually long periods. In particular, at maximum activation by hyperpolarization and saturating cAMP, the open probability approaches unity. In contrast to other reports, no evidence of interchannel cooperativity was observed. In conclusion, single HCN2 channels operate only with an exceptionally low conductance, and both activating stimuli, voltage and cAMP, exclusively control the open probability.     

Einfluß von D,L-Ethionin auf Zellkulturen von Ruta graveolens L

D,L-Ethionine was added in varying concentrations (0.1–1 mM) to two cell suspension cultures of Ruta graveolens. Growth, alkaloid formation and activities of some shikimate pathway-specific enzymes in these cultures were estimated. Also the effect of ethionine on shikimate pathwayspecific enzymes under in vitro conditions was followed. Growth is only slightly inhibited in supplemented cultures. Alkaloid formation is drastically reduced in a low-producing and to a lesser extent in the high producing cell line by ethionine. Activities of DAHP synthase, chorismate mutase, and anthranilate synthase in the presence of ethionine are in different Ruta strains to a varying degree affected.     

Microbiology in the 1990s: reflections about open questions and trends

The optimal conditions for protoplast formation ofCandida apicola were by using an enzyme fromArthrobacter sp. in combination with 2-mercaptoethanol. The kinetic data support the two-layered structure model of cell wall for this yeast but the structure of the cell wall depended on the age of cells and culture conditions. To regenerate the protoplasts, the type of osmotic stabilizer was important: sorbitol gave 16 to 30% regeneration. Electron microscopy revealed the presence of vesicles in the sections of protoplasts and whole cells ofCandida apicola grown in production medium and producing glycolipids. In sections of whole cells, vesicle-like structures are located in the periplasmic space and in protoplasts they can either be attached to, or released from, the cell surface. These vesicles are thought to be involved in the transport of the surface-active glycolipids and in the protection of the cell against denaturing effects.     

Immobilization ofPenicillium raistrickii spores and mycelium growth for 15α-hydroxylation of 13-ethylgon-4-en-3,17-dione

Spores ofPenicillium raistrickii immobilized by gel entrapment, microencapsulation and surface adsorption were allowed to grow into mycelial form, in order to accomplish hydroxylation of 13-ethylgon-4-en-3,17-dione at the 15α-position. The hydroxylating activity of both free and immobilized cells at different pH and substrate concentration as well as under starvation conditions was studied. The productivity of cells immobilized by several techniques is compared.