Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

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.     

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.     

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.