An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.     

TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae.

Incubation of Saccharomyces cerevisiae with the plant cytokinin N6-(delta 2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures. Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol. This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation. However, measurement of stress-induced thermotolerance in two mutant strains (tpk1, tpk2, TPK3; tpk1, TPK2, tpk3) each deficient in two of the catalytic subunits of the cAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK. In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression. Therefore, this implies there must be an alternative novel mechanism, other than cAMP, for activating cAPK during stress. Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance. However, incubation in 2iP or ethanol had no significant effect. This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms. Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels.     

Activation of Guanylate Cyclase by Bradykinin in Rat Sensory Neurones Is Mediated by Calcium Influx: Possible Role of the Increase in Cyclic GMP

Bradykinin, which activates polymodal nociceptors, increased cyclic GMP (cGMP) in a capsaicin-sensitive population of cultured sensory neurones from rat dorsal root ganglia (DRG) by stimulating guanylate cyclase, but had no effect on cyclic AMP (cAMP). In nonneuronal cells from DRG, bradykinin increased cAMP, but not cGMP. The bradykinin-induced increase in cGMP in the neurones was completely blocked by removal of extracellular Ca2+, or by incubation of the cells with the calcium channel blockers nifedipine and verapamil. Pretreatment of the neurones with either dibutyryl cGMP or sodium nitroprusside (which elevates cGMP) inhibited bradykinin-induced formation of inositol phosphates. It is possible that cGMP could be involved in the regulation of polyphosphoinositide turnover in DRG neurones.