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.     

Secondary conformational polymorphism of nucleic acids as a possible functional link between cellular parameters and DNA packaging processes

Circular dichroism and electron microscopy studies of various in vitro DNA packaging systems indicate that all the factors which induce and modulate the secondary conformation of DNA molecules are capable of eliciting nucleic acids condensation processes into tight, highly ordered tertiary structures as well as altering the extent of order and compactness within the resulting species. Specifically, such factors include the ionic strength, the presence of particular dehydrating agents and polyamines, as well as the pH values. It is proposed that slight alterations of these parameters induce the formation of short non-B-DNA segments that propagate as a perturbation along the B-DNA double helix. The structural fluctuations of the dsDNA molecules that result from the conformational discontinuities formed at the junction sites between the B motif and the conformationally altered segments alter the elastic response of the nucleic acids and facilitate cooperative condensation processes. Moreover, the type and frequency of the structurally modified clusters interspersed within the B conformation and determined by the environmental parameters are shown to provide a means for continuous regulation of the extent and mode of DNA packaging. The ionic strength and hydrophobic environment in the close vicinity of the DNA molecules are controlled and modulated in vivo by DNA-binding proteins such as histones and protamines; similarly, pH values and polyamine concentrations are constantly regulated in living systems. It is suggested, therefore, that the secondary structural polymorphism which characterizes the DNA molecules might display a regulatory role by acting as a functional link between cellular parameters and the extent, mode, and timing of nucleic acid packaging processes.     

Specific inactivation of hepatic fatty acid hydroxylases by acetylenic fatty acids

The terminal acetylenic analogue of lauric acid, 11-dodecynoic acid (11-DDYA), specifically inactivates hepatic cytochrome P-450 enzymes that catalyze omega- and omega-1-hydroxylation of lauric acid. The inactivation, as required for a suicidal process, is NADPH- and time-dependent and follows pseudo-first order kinetics. In contrast, 11-DDYA causes no measurable change in the spectroscopically-measured concentration of cytochrome P-450 or in the N-demethylation of benzphetamine or N-methyl p-chloroaniline. 10-Undecynoic acid is as effective a suicide substrate for fatty acid hydroxylases as 11-DDYA but 11-dodecenoic acid is much less effective. 11-DDYA is able to completely inhibit omega-hydroxylation but suppresses no more than 50% of omega-1-hydroxylation despite the fact that both activities are completely inactivated by 1-aminobenzotriazole. At least three hepatic cytochrome P-450 fatty acid hydroxylases, one omega-hydroxylase and two omega-1-hydroxylases, are required by these results. The construction of suicide substrates that specifically inactivate cytochrome P-450 fatty acid hydroxylases provides a new experimental probe of the physiological role of this process.     

The carbon-13 nuclear magnetic resonance spectra of four eudesmane sesquiterpenols

The ¹³C NMR spectra of geosmin, selina-4(14),7(11)-diene-99-ol and two dihydroeudesmol isomers have been obtained and the individual resonances assigned. Several different empirical correlations developed by others have been combined in simple calculations to predict chemical shift values for sesquiterpenols of the eudesmane group.