Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Stimulus-evoked slow potential shifts and changes in [K+]0 of the frog optic tectum

Summary In 17 frogs (Rana esculenta var ridibunda) immobilised with succinyl choline the optic tectal surface was stimulated by trains of electrical pulses or by a flash to the contralateral eye. Sustained potential shifts (SPSs) and changes in extracellular potassium concentration ( [K⁺]₀) were simultaneously recorded.In response to electrical stimulation SPSs of maximal amplitudes (1.19±0.1 mV) were recorded between 50 and 200 m in depth and maximal [K⁺]₀ (0.69 ±0.08 mM) between 100 and 350 m. The changes of SPS and [K⁺]₀ showed a close similarity in experiments with changes in voltage, pulse duration and frequency of stimuli within a train. The induced SPS had a duration of 28±1.54 s, the [K⁺]₀ of 32±1.23 s.The flash stimulus induced an SPS and [K⁺]₀ of maximal amplitudes between 50 and 200 m in depth with values of 0.57±0.1 mV and 0.29±0.03 mM respectively. An additional wave with a latency of ca 1 s and a duration of ca 3 s arose on the background of the SPS to a flash stimulus, associated with an additional increase in [K⁺]₀.It is considered that the accumulation of K⁺ in extra-cellular space, with neuronal activity, results in depolarization of radial processes of ependymal glia. This is reflected in the neuropil of the upper layers of the optic tectum as an SPS.We would like to dedicate this article to the memory of Alexander Roitbak who died as a result of a tragic accident while this paper was in press. He will be remembered fondly especially for his contributions to understanding of the functions of Neuroglia. E.V.O., P.R.L., T.A.R.     

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.