An emerging pharmacology of peptide toxins targeted against potassium channels

Summary Voltage-dependent ion channels are a difficult class of proteins to approach biochemically. Many such channels are present at low density in relevant tissues and exist as multiple subtypes that can be distinguished electrophysiologically. In particular, K channels appear to be a diverse family of proteins characterized by many different conductance properties, gating behaviors and regulatory phenomena. Fortunately, specific peptide toxins for K channels are present in the venoms of insects, scorpions, snakes and possibly other species. The available sequences of these peptides define several different families of toxins. Electrophysiological and radioligand binding studies suggest that these toxins can be used to distinguish subclasses of K channels that share similar toxin binding sites. The growing databank of sequence homologies for both toxins and channels is, in essence, a codebook for identifying common elements of structure and function. The continuing development of toxins as biochemical probes should help to uncover the molecular basis and physiological significance of K-channel diversity.     

Complementation by intraspecific protoplast fusion of auxotrophic cell lines of Datura innoxia P. Mill.

It was determined using electrophoresis in polyacrylamide gels containing native DNA or RNA that sugar non-specific nuclease active at pH 5.2 was expressed in tobacco callus. The nuclease had a relative molecular mass of about 34.6 kDaltons and degraded substrates in the following order of decreasing rate: denaturated DNA>poly dA>UV-irradiated native DNA>native DNA>alkylated native DNA>apurinated native DNA>poly dGpoly dC. The nuclease activity changed during callus growth and plant regeneration, but no developmental changes in electrophoretic patterns were detected. The increase in specific DNAse activity of nuclease was maximal in the exponential phase of callus growth on both growth and regeneration media, except for activity in the cytokinin-independent cell strain grown on growth medium. The specific DNAse activity of nuclease decreased during the bud formation period, while total DNAse activity calculated per mg of dry weight was slightly higher in vegetative buds (9.1U) than in undifferentiated tissue of callus (8.5U). Specific DNAse activity was, on the average, several hundred-fold lower in the vegetative tissues of flowering tobacco plants than in calluses in the exponential phase of growth.     

Somatic embryogenesis and plant regeneration from zygotic embryo culture in blue spruce (Picea pungens Engelman.)

Summary Somatic embryogenesis and plantlet formation have been achieved from cultured mature zygotic embryos of blue spruce (Picea pungens Engelman.). The effect of three basal media LP, LM, and BLG, all used at half-strength, was tested at the induction phase. LM medium induced somatic embryogenesis to a higher extent than LP whereas BLG did not produce any embryonal-suspensor mass representing stage 1 somatic embryos. The embryonal-suspensor mass was induced on a wide range of auxin/cytokinin ratios. However, media containing either 2 M NAA and 10 M BA, or 10 M NAA and 5 M BA produced somatic embryos that gave the highest frequency of plantlets. The level of ABA required in the maturation medium for somatic embryos to mature properly varied with the auxin/cytokinin levels in the induction medium on which the somatic embryos were derived. Inclusion of AgNO₃ (10 – 100 M) in the induction medium reduced somatic embryogenesis and embryo conversion.Abbreviations NAA naphthalene-acetic acid - BA N⁶-benzylaminopurine - ABA abscisic acid     

Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth regulator levels and significance of cotyledons

The regulatory role of thidiazuron (TDZ) and explant factors in imparting somatic embryogenic potential was assessed in relation to endogenous growth regulator levels in peanut ( Arachis hypogaea L. cv. Tango). TDZ induced somatic embryogenesis over a range of concentrations (0.5 to 10 μ M ). Culture of seedlings for just 2 days on TDZ-supplemented medium was sufficient to induce somatic embryogenesis. Seedling age and retention or removal of cotyledons during culture influenced morphogenic potential significantly. The younger the seedlings, the better was the embryogenic response to TDZ treatment. The embryogenic potential of seedlings was limited for explants with no cotyledons and they did not respond to increasing levels of TDZ. In contrast, retention of at least one or both the cotyledons resulted in increased response to TDZ. Endogenous levels of cytokinins, auxins and abscisic acid were influenced by TDZ treatment, while TDZ itself was detected only in the cotyledons. The cytokinin N⁶-(2-iso-pentenyl)adenine (2iP) fluctuated on a 10-day cycle in the cotyledons; TDZ treatment suppressed this change and caused an overall decrease in the pool of available 2iP in the tissue. It appeared that TDZ induced somatic embryogenesis in peanut by influencing endogenous levels of both auxin and cytokinins.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Development of gametophores from isolated protoplasts of the mossAnoectangium thomsvnii Mitt.

Summary Protoplasts of the mossAnoectangium thomsonii Mitt. were isolated from preplasmolyzed protonemal filaments, grown in suspension cultures, after the digestion of the cell wall by the enzymes cellulase and macerozyme or driselase. Driselase was more effective than cellulase and macerozyme. After purification these protoplasts were plated in the form of small agar drops in modified Kofler's medium without hormones and incubated in the dark at 26 ± 2 °C. Cell walls regenerated within three days and cell divisions started seven days after the initiation of the cultures. When the regenerants were transferred to normal protonemal culture medium and illuminated by 3,000 lux continuous light, a multi-cellular protonema developed which formed leafy gametophores on salicylic acid supplemented medium.     

Thidiazuron: A potent regulator ofin vitro plant morphogenesis

Summary TDZ (N-phenyl-N’-1,2,3-thidiazol-5-ylurea) is a substituted phenylurea compound which was developed for mechanized harvesting of cotton bolls and has now emerged as a highly efficacious bioregulant of morphogenesis in the tissue culture of many plant species. Application of TDZ induces a diverse array of cultural responses ranging from induction of callus to formation of somatic embryos. TDZ exhibits the unique property of mimicking both auxin and cytokinin effects on growth and differentiation of cultured explants, although structurally it is different from either auxins or purine-based cytokinins. A number of physiological and biochemical events in cells are likely to be influenced by TDZ, but these may or may not be directly related to the induction of morphogenic responses, and hence, the mode of action of TDZ is unknown. However, the recent approaches applied to study the morphogenic events initiated by TDZ are clearly beginning to reveal the details of a variety of underlying mechanisms. Various reports indicate that TDZ may act through modulation of the endogenous plant growth regulators, either directly or as a result of induced stress. The other possibilities include the modification in cell membranes, energy levels, nutrient uptake, or nutrient assimilation. In this review, several of these possiblities are presented and discussed in light of recently published studies on characterization of TDZ-induced morphogenic effects.     

Requirement of Ca2+ for aflatoxin production: inhibitory effect of Ca2+ channel blockers on aflatoxin production by Aspergillus parasiticus NRRL 2999

Aflatoxin production by Aspergillus parasiticus NRRL 2999 was inhibited when Ca2+ channel blockers, i.e., verapamil and diltiazem (> 1 mmol 1(-1)), were included in the culture medium. Inhibition was not accompanied by growth inhibition, nor was the [14C]-glucose uptake by the organism altered. However, both the compounds inhibited [14C]-acetate incorporation into aflatoxin B1 in a dose-dependent manner and decreased sporulation of the organism. Even though a nutritional role for Ca2+ has not been demonstrated unequivocally in fungi, the present study suggests the importance of Ca2+ in the production of these secondary metabolites.     

High-frequency Organogenesis from Direct Seed Culture in Lathyrus

Culture conditions were developed for inducing a high frequencyof direct shoot morphogenesis and whole plant regeneration incultures of intact seedlings of Lathyrus cicera L., L. ochrus(L.) DC., L. sativus L., and L. tingitanus L. The procedureof shoot regeneration involved culturing of whole, mature seedson MS medium containing cytokinins, or thidiazuron (TDZ), asubstituted phenylurea with cytokinin-like activity. Differentiationof shoots occurred without an intervening callus phase fromthe cotyledonary node and surrounding tissues of the intactseedlings developed from seeds germinated on media containingkinetin, BAP or TDZ. An average of 19·0, 15·8,28·8 and 43·0 shoots were regenerated at optimalconcentrations of 50·0, 50·0 and 80·0 µMBAP in L. cicera, L. tingitanus, L. ochrus and L. sativus, respectively.TDZ enhanced the shoot formation at the concentration of 10µM to an average of 33·1 and 33·7 shootsper seedling in L. cicera and L. tingitanus and at 50 µM,to 57·4 shoots per seedling in L. sativus. Regeneratedshoots developed roots on a modified MS medium containing 2·5µM NAA and the surviving plantlets were transferred tosoil. Histological studies revealed de novo formation of shoot budsfrom the epidermal or subepidermal cells of the basal and nodalregion of multiple epicotyls. Several meristematic centres consistingof actively-dividing cells developed in the subepidermal celllayer of the nodal tissue and adjacent areas within 7 d of seedculture on a cytokinin- or TDZ-supplemented medium. The patternof the development of these meristematic centres and shoot developmentwas similar in all four species.Copyright 1993, 1999 AcademicPress Seed culture, direct shoot morphogenesis, Lathyrus, thidiazuron, grass pea, vetch ochrus