In vitro differentiation of rabbit blastocyst cells

Summary Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was −59±1.2 mV and the threshold potential was −53±2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect. This work was supported by grants HD21302, HD07069, DK31091, and HL37320 from the National Institutes of Health, Bethesda, MD, with additional support from a University of Alabama at Birmingham Cardviovascular Research and Training Center Award.     

The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.     

Two-step purification of full-length nerve growth factor receptor and maintenance of receptor-specific binding activity

A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

H-2T24 and Pema T24: orthologous expressed MHC class Ib genes from mouse and Peromyscus maniculatus

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U03104 and L22338.     

The biogeochemistry of a north-temperate grassland with native ungulates: Nitrogen dynamics in Yellowstone National Park

Nutrient dynamics of large grassland ecosystems possessing abundant migratory grazers are poorly understood. We examined N cycling on the northern winter range of Yellowstone National Park, home for large herds of free-roaming elk (Cervus elaphus) and bison (Bison bison). Plant and soil N, net N mineralization, and the deposition of ungulate fecal-N were measured at five sites, a ridgetop, mid-slope bench, steep slope, valley-bottom bench, and riparian area, within a watershed from May, 1991 to April, 1992.Results indicated similarities between biogeochemical properties of Yellowstone grassland and other grassland ecosystems: (1) landscape position and soil water affected nutrient dynamics, (2) annual mineralization was positively related to soil N content, and (3) the proportion of soil N mineralized during the year was negatively related to soil C/N.Grazers were a particularly important component of the N budget of this grassland. Estimated rates of N flow from ungulates to the soil ranged from 8.1 to 45.6 kg/ha/yr at the sites (average = 27.0 kg/ha/yr), approximately 4.5 times the amount of N in senescent plants. Rates of nitrogen mineralization for Yellowstone northern range grassland were higher than those measured in other temperate grassland ecosystems, possibly due to grazers promoting N cycling in Yellowstone.