After initial resuscitation from human septic shock, a single dose of methylene blue transiently increases mean arterial pressure and oxygen uptake, associated with a decrease in arterial compliance and increases in myocardial function and oxygen delivery. Hence, nitric oxide may be a mediator of the circulatory changes of human septic shock.

Shifting a yeast culture from an ethanol-based medium to a glucose-based medium causes a coordinate increase of the cellular levels of ribosomal protein mRNAs by about a factor 4 within 30 min. Making use of hybrid genes encompassing different portions of the 5'-flanking region of the L25-gene, we could show that the increase in mRNAs is a transcriptional event, mediated through DNA sequences upstream of the ribosomal protein (rp) genes. Further analysis revealed that sequence elements are involved that many rp-genes have in common and that previously were identified as transcription activation sites (RPG-boxes or UASrpg). Using appropriate deletion mutants of the fusion genes we could demonstrate that a single RPG-box is sufficient for the transcriptional upshift. In addition, both copy genes encoding rp28 which differ considerably in their extent of transcriptional activity, show the upshift effect in a proportional manner. Definite proof for the role of the UASrpg in nutritional regulation was obtained by examining the effect of a synthetic RPG-box on transcription.

What is the highest possible replication rate for living organisms? The cellular growth rate is controlled by a variety of processes. Therefore, it is unclear which metabolic process or group of processes should be activated to increase growth rate. An organism that is already growing fast may already have optimized through evolution all processes that could be optimized readily, but may be confronted with a more generic limitation. Here we introduce a method called ‘cell selection’ to select for highest growth rate, and show how such a cellular site of ‘growth control’ was identified. By applying pH‐auxostat cultivation to the already fast‐growing yeast Kluyveromyces marxianus for a sufficiently long time, we selected a strain with a 30% increased growth rate; its cell‐cycle time decreased to 52 min, much below that reported to date for any eukaryote. The increase in growth rate was accompanied by a 40% increase in cell surface at a fairly constant cell volume. We show how the increase in growth rate can be explained by a dominant (80%) limitation of growth by the group of membrane processes (a 0.7% increase of specific growth rate to a 1% increase in membrane surface area). Simultaneous activation of membrane processes may be what is required to accelerate growth of the fastest‐growing form of eukaryotic life to growth rates that are even faster, and may be of potential interest for single‐cell protein production in industrial ‘White’ biotechnology processes.

No abstract

The physiological consequences of overproduction of the homologous glycolytic enzyme 3-phosphoglycerate kinase (PGK), integrated in 80 PGK1 gene copies in the genome of Saccharomyces cerevisiae are described. This multiple integration and the strong PGK overproduction (maximum 47% of the total soluble cell protein) do not affect the maximal specific growth rate, but cause 40% reduction of the molar growth yield, compared with that of the wild-type host. The extra energy that is needed for protein overproduction is mainly provided by extra fermentation (respirofermentative growth), but respiration is also elevated compared with the reference strains. The increase in the specific oxygen uptake rate indicates that the respiratory capacity of the yeasts is higher than that in the wild-type host, in which the limited capacity of respiration is generally supposed to be at its maximal level at the critical dilution rate, and is thus responsible for the switch to respirofermentative growth. In a medium PGK1 gene copy integrant (about 25 copies), overproduction of 10%-12% PGK has a stimulating effect on the growth yield and energy efficiency. In these cells the growth benefits of overproduction of the glycolytic enzyme are higher than the disadvantages of extra protein synthesis. The overproduction of PGK has also consequences for the glucose affinity of the yeasts: In the more overproducing strain the Ks is increased, compared to its reference strains. Elimination of strong overproducing cells from a glucose-limited chemostat culture is caused by two factors: (a) the excision of the PGK genes from the genome, which is of minor importance for wash-out, but the induction process for this overall decline of overproduction, and (b) the physiolOffprint requests to: P. C. van der Aar ogical selection process for less overproducing cells, caused by differences in affinity for glucose, most obvious at # ~, l/2#max. However in batch culture and in a chemostat at low specific growth rates, all the overproducing strains show high genetic stability and constantly provide high PGK quantities.