Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

GENETIC RELATEDNESS OF TWO POPULATIONS OF THE SOUTHERN ELEPHANT SEAL, MIROUNGA LEONINA

Blood samples from southern elephant seals ( Mirounga leonina ) from Heard and Macquarie Islands were surveyed electrophoretically for protein variation. Thirty proteins encoded by a minimum of 35 loci were screened, four of which were found to be polymorphic. Statistically significant differences in allele frequencies were found between the two populations at three loci. Heterozygosity estimates for the Heard and Macquarie island populations were 0.034 ± 0.020 (mean ± standard error) and O.029 ± 0.017 respectively, with a Nei distance of 0.007. The findings suggest that the two populations may have diverged genetically and very limited gene flow exists between the islands, a finding consistent with limited information from mark-recapture studies.     

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.     

Reaction on alternating GC oligonucleotide templates

Summary The condensation reactions of activated nucleotides, ImpN or 2-MeImpN, with the selfcomplementary ribo-octanucleotide GCGCGCGC or with the partially self-complementary heptanucleotide GCGCGCG were studied. The templatedirected reaction of 2-meImpC with the heptamer yields the 3–5 octamer as the main product. All other reactions yield 2–5-linked octamers and pyrophosphates as major products. Surprisingly, 2-MeImpG facilitates the reaction of 2-MeImpC with the heptamer.Procedures for the analysis by gel electrophoresis of the oligomeric products obtained in reactions of this kind are described.     

Two-dimensional gel electrophoresis as a taxonomic tool: Evidence from the centrospermae

Two-dimensional polyacrylamide gel (2-D PAGE) electrophoresis was appraised as an experimental technique for assessing systematic relationships among higher plants and to determine at which level in the taxonomic hierarchy this technique is most generally applicable. 2-D PAGE was performed on denatured extracts of mature leaves from 25 species representing five families of the order Centrospermae (Caryophyllales, Chenopodiales) in the Angiospermae as well as Welwitschia mirabilis (Gymnospermae). Cluster analysis of a 256 spot binary-coded data set derived from the computer-encoded spot patterns of the 25 species successfully separated taxa from the individual to the familial levels of the taxonomic hierarchy in accordance with traditional taxonomic delineations of the taxa.     

Properties of cyanogen bromide-activated, Agarose-immobilized catechol 1,2-dioxygenase from freeze-dried extracts of Nocardia sp. NCIB 10503

Catechol 1,2-dioxygenase [catechol: oxygen 1,2-oxidoreductase (decyclizing); EC 1.13.11.1], the aromatic intradiol ring-cleaving enzyme of Nocardia sp. NCIB 10503 prepared by freeze-drying cell-free extracts, was covalently attached to cyanogen bromide-activated Agarose. The properties of the immobilized enzyme were compared to those of the free enzyme preparation. Immobilization was shown to increase the thermal stability of the enzyme. The pH-activity profile was altered by immobilization. Various explanations for this phenomenon are discussed. The V_max and K_m of the enzyme were not significantly affected on immobilization. The enzyme had a broader substrate specificity than any previously reported catechol 1,2-dioxygenase, and this was largely unaltered by immobilization. The properties of the preparations are compared to those of other (free) catechol 1,2-dioxygenases. The results presented show that the immobilization of catechol 1,2-dioxygenase offers an attractive means for the production of cis,cis-muconate and novel substituted analogues.     

Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of differentiated syrian golden hamster pancreatic duct cells maintained in extended monolayer culture

Summary Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium suppleneted with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal growth factor, 3,3′, 5-triodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to bovine serum albumin (ITS⁺), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous elongated mitochondria, and both free and membrane-bound ribosomes. Cell grown as monolayers for 3 mo. were more flattened and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic AMP (cAMP) after stimulation by 1×10⁻⁶ M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold. At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels of cAMP after exposure to serretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic studies. This research was supported by grant CA34051 from the National Cancer Institute, Bethesda, MD.     

Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.