A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

Expression of renal cell protein markers is dependent on initial mechanical culture conditions.

The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.     

Ethylene production by axenic fruiting cultures of Agaricus bisporus

Axenic cultures of Agaricus bisporus were used to show that the rise in ethylene production during fruiting derives from its own metabolism.     

Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K_m for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a K_m for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.     

Differences among immune complexes: association of C1q in SLE immune complexes with renal disease

Studies that made use of multiple assay systems demonstrated increased levels of immune complexes (IC) in patients with systemic lupus erythematosus (SLE), but no consistent correlations of IC concentration to patterns or activity of disease have been observed. Furthermore, consistent associations between qualitative differences in IC and disease manifestations have been elusive. IC interaction with erythrocytes and mononuclear phagocytic cells is another variable in SLE that may also mediate some of the biological effects of IC. The present report concerns studies of the composition of purified IC obtained from individuals with SLE and other rheumatic diseases; a 64,000 dalton component identified as the A-B subunit of C1q was detected in purified IC from 27 of 51 SLE patients (53%). The presence of this 64,000 dalton component was not related to either IC concentration or to the serum C1q level. However, the presence of the C1q component in isolated SLE IC did correlate with the presence of renal disease (p less than 0.02). These observations are interpreted relative to a recently described kinetic model of IC clearance.     

Application of the Fröhlich theory to the modelling of rouleau formation in human erythrocytes

The details of Fröhlich's theory and some recent experiments on the rouleau formation of human erythrocytes which exhibit a strong interaction that appears to satisfy the prerequisites of the Fröhlich theory, are summarized. To verify whether the Fröhlich theory of long-range coherence in biological systems is applicable to the phenomenon of rouleau formation in human erythrocytes, the interactions between erythrocytes are modelled as those between two large, coupled oscillating dipoles. Relevant expressions for the resonant long-range and the van der Waals interaction are then derived. Using the available numerical data, the eigenfrequencies and the interaction energies corresponding to the experimental conditions are then derived. In the range of postulated frequencies (10¹¹–10¹² Hz) the effective interaction coefficient due to the resonant long-range forces is, indeed, found to agree with its experimental value of 3.0. However, the same value of can also be achieved through the ordinary van der Waals interactions between dipoles oscillating at lower frequencies. It is concluded that the resonant long-range interaction between erythrocytes may be responsible for the onset of rouleau formation. However, other mechanisms cannot be ruled out at this stage, especially since the Fröhlich mechanism requires a number of unconfirmed preconditions.