Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. "Leaky" cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.     

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.     

Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme

Sequential deletion of the carboxyl-terminal amino acids (including the six direct repeating units) of the glucosyltransferase-I (GTF-I) enzyme of Streptococcus mutans revealed differential effects on sucrase and GTF activities. Removal of all but one repeating unit resulted in a truncated enzyme with significant sucrase activity but no detectable GTF activity. These results are compatible with the presence of two functional domains in the enzyme.     

Establishment of a transient expression system for Dictyostelium discoideum.

We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes.     

Visual optics in toads (Bufo americanus)

Aspects of visual optics were investigated in the American toad (Bufo americanus). The development of the refractive state of the eye during metamorphosis was followed with IR photoretinoscopy. Frozen sections documented the changes in optical parameters before and after metamorphosis. There is a difference in light sensitivity between juvenile and adult toads. Binocular accommodation in adult toads was observed. 1. IR photoretinoscopic measurements showed that the refractive state of the eye changed very rapidly during metamorphosis, about 10 D/h while the animal entered the terrestrial habitat. 2. Frozen sections showed that the almost spherical lens in a tadpole eye had flattened in a just metamorphosed toad's eye while at the same time the distance of the lens to the retina had decreased. However, the morphological measurements were not sufficiently sensitive to record the relatively small changes in ocular dimensions that were responsible for the rapid changes in refractive state during metamorphosis. 3. Schematic eyes, with homogeneous and non homogeneous lenses, were constructed for tadpoles, juvenile toads, and adult toads. 4. Nonparaxial raytracing studies in schematic eyes suggested that the lenses of animals of the three developmental stages tadpole, juvenile toad, and adult are not homogeneous but have a refractive index gradient. The raytracing studies indicated that the refractive index gradient is different for the different developmental stages, being highest in the tadpole lens. 5. The observations of toads during feeding behavior at different light levels showed an increased light sensitivity in the adult nocturnal toads in contrast to the juvenile animals, which are diurnal. The increased light sensitivity could partly be explained with an increase in aperture and an increase in red rod outer segments. To fully explain the higher light sensitivity in adult toads, changes in neuronal parameters had to be assumed. 6. Retinoscopic measurements of the resting refractive state in the adult toad showed a hyperopic defocus of about +8 D. By subtracting the measurement artefact for retinoscopy, the true resting focus was found to be nearly emmetropic. 7. The amount of natural accommodation in adult toads during normal feeding behavior was investigated with IR photoretinoscopy. Binocular accommodation of about 8 D was observed.     

Lack of cardiovascular tolerance during intravenous cocaine infusions in human volunteers

Acute tolerance to the cardiovascular effects of cocaine has been hypothesized from experiments in which the plasma concentrations of cocaine were rapidly changing. We studied the cardiovascular responses of 8 male human subjects for 4 hours following intravenous bolus doses of cocaine, and compared these to responses in the same subjects after intravenous bolus doses of cocaine followed by continuous intravenous infusions of cocaine designed to maintain steady state plasma levels of cocaine. We found little evidence of tolerance to the tachycardia and hypertensive effects of cocaine during a four hour exposure. Lack of tolerance to the cardiovascular effects of cocaine may be a factor in some types of cocaine related toxicity among cocaine abusers.