Biomedical subjects
Publications and source records attributed to J Tsao.
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A series of transformants have been derived which carry the Col plasmids from 11 E-colicin-producing strains isolated by Fredericq or Hamon. The ColE plasmids identified included four of type E1, five of type E2, and two of a type designated E4 by Horak (Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 233:58-63, 1975). Strain K317 was shown to carry a ColE7 plasmid and a 2.7-megadalton (Md) ColE2imm plasmid which confers immunity to colicin E2 but not the ability to produce colicin. Other plasmids identified in the colicinogenic isolates were a 3.4-Md ColN plasmid and a 1.3-Md Col plasmid of unknown type. The ColE1 plasmids all continued replicating in cultures treated with chloramphenicol. Strains carrying the ColE4 or ColE3-CA38 plasmid exhibited partial sensitivity to their own colicins and exhibited an unusual clearing-zone morphology when overlaid on stabs of colicin E2- or E7-producing strains. Except for ColE1-K53, ColE1-K47, ColE2-CA42, and ColE7-K317, all of the ColE plasmids were found to be about 4.3 Md in size. ColE2-CA42 and ColE7-K317 are both about 3.9 Md and are the only two plasmids of the E2, E3, E4, and E7 types that did not yield a 0.39-Md deoxyribonucleic acid fragment as an EcoRI digestion product. The comparisons of the ColE plasmids suggest several structural and functional relationships among them.
Two groups of mutant clones were isolated from YI adrenocortical tumor cells. One group, Y1(Kin), exhibited altered cytosolic cyclic AMP-dependent protein kinase activity; the second group, Y1(Cyc), exhibited diminished corticotropin-responsive adenylate cyclase activity. Steroidogenic responses to corticotropin and cyclic nucleotides closely paralleled cyclic AMP-dependent protein kinase activity in the Y1(Kin) mutants. In Y1(Cyc) mutants, corticotropin had little effect on steroidogenesis, whereas cyclic nucleotides were fully active. These data imply that adenylate cyclase and cyclic AMP-dependent protein kinase are obligatory components of the corticotropin-stimulated steroidogenic pathway.
Two variant cell lines (Y6 and OS3), derived from the ACTH-sensitive mouse adrenocortical tumor clone Y1, are defective in the ACTH-sensitive adenylate cyclase system. This study further characterizes the nature of the defects in Y6 and OS3 cells using ACTH1-10, ACTH4-10, and cholera toxin. In Y1 cells, ACTH1-39, ACTH1-10, and ACTH4-10 stimulated steroidogenesis to the same maximum level with Kd' values of 5 x 10(-11) M, 5 x 10(-7) M and 10(-4) m respectively. ACTH1-10 (0.4 mM) and ACTH4-10 (3.2 mM) increased the accumulation of adenosine 3',5'-monophosphate (cAMP) in Y1 cells two- to three-fold. Cholera toxin increased steroidogenesis and cAMP accumulation in Y1 cells with Kd' values of 0.4 ng/mL and 9 ng/mL respectively. Y6 and OS3 cells responded to added cholera toxin with increased cAMP accumulation and increased steroidogenesis but did not respond to ACTH1-39, ACTH1-10, or ACTH4-10 at concentrations effective in Y1 cells. These data are interpreted to suggest that Y6 and OS3 cells are defective in a process or component that links the principal binding regions of the ACTH receptor to the catalytic subunit of the adenylate cyclase system. Attempts to were made to assess the interactions of ACTH with the principal binding regions of the ACTH receptor by analysis of binding of radioactive, iodinated ACTH1-24. ACTH binding, however, showed low affinity, high capacity, and no target-tissue specificity, and was considered not to be useful in evaluating the integrity of the ACTH receptor.
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A somatic cell genetic approach was used to study the role of cyclic nucleotides in adrenal steroidogenesis. 8-Bromoadenosine 3',5'-monophosphate (8BrcAMP) stimulated steroidogenesis (K'd=0.1 mM) in cultured mouse adrenocortical tumor cells (Clone Y1). In addition, 8BrcAMP inhibited Y1 cell growth and caused Y1 cell monolayers to assume a rounded morphology. As a consequence, 8BrcAMP (at concentrations greater than or equal to 0.4 mM) reduced the relative plating efficiency of Y1 cells to less than 10(-5). Y1 cells were mutagenized with ethyl methanesulfonate (300 microgram/ml) and grown in the presence of 0.4 mM 8BrcAMP. A surviving colony (8BrcAMPr-1) was shown to be resistant to growth inhibition (relative plating efficiency at 1.0 mM 8BrcAMP=50 percent)) and to morphological changes induced by 8BrcAMP. 8BrcAMPr-1 cells had diminished steroidogenic responses to cyclic nucleotides and to ACTH (less than or equal to 33 percent of maximum). In 8BrcAMP(R)-1 cells, adenylate cyclase activity remained responsive to ACTH, and cyclic AMP phosphodiesterase activity was not increased. These data suggest that 8BrcAMPr-1 cells are defective at a point common to cyclic AMP action on growth, morphology and steroidogenesis. The associated decrease in responsiveness of the steroidogenic pathway to ACTH suggests that ACTH-regulated steroidogenesis is via a cyclic nucleotide-mediated mechanism.
The Helene Fuld Leadership Initiative in Nursing Education (LINE) program was designed to enhance beginning leadership competencies of baccalaureate nursing students. Given the increasing need for strong and effective leadership throughout the health care system, and the demands new graduates encounter as they move into practice, the LINE program is built on the premise that leadership skills must be instilled at the undergraduate level. The program achieves its goal through an intensive 5-day institute focused on assessing and developing the leadership competencies of nurse educators and their clinical partners to enable them to be effective agents of curriculum change in their home institutions. The institute also assists participants to redesign their baccalaureate nursing (BSN) curricula to ensure that students learn to: (1) work effectively within and across complex, integrated organizational and institutional boundaries; (2) think and act from the perspective of a system; and (3) communicate, negotiate, lead, and facilitate change within health care organizations. D. Goleman's (1998) framework of emotional intelligence, which addresses both personal competence (managing oneself) and social competence (handling one's relationships with others) provides the framework for operationalizing leadership in the BSN curriculum. To date, 26 BSN programs and their clinical partners have participated in the LINE program, which has the potential to influence the beginning leadership development of more than 2,400 BSN students. Program outcomes reveal that education-practice collaboration, professional networking, individual leadership development of nurse educators and their clinical partners as change agents, and the integration of leadership experiences at all levels of the BSN curriculum are important in developing beginning leadership competencies in BSN students.
Forskolin reduced the plating efficiency of Y1 adrenocortical tumor cells in a concentration-dependent manner--more than 5-orders of magnitude at 10 uM forskolin and at least 6-orders of magnitude at 50 uM forskolin. This effect was related to the diterpene's ability to increase adenylate cyclase activity and adenosine 3',5'-monophosphate (cAMP) levels in Y1 cells. Stable, forskolin-resistant mutants were isolated following growth of Y1 cells for 3 to 4 weeks in the presence of 10 uM forskolin. These mutants were stable, were present in the population at a ratio of approximately 15 mutants per million cells and appeared to result from a defect in cAMP accumulation rather than cAMP action. The forskolin-resistant phenotype was associated with a reduced ability of forskolin to stimulate adenylate cyclase activity in intact cells and in cell homogenates. The adenylate cyclase system of forskolin-resistant mutants was responsive to NaF, but was virtually insensitive to corticotropin (ACTH). As determined by a modified fluctuation analysis, the forskolin-resistant phenotype arose by spontaneous mutation at a frequency consistent with a mutational event at a single genetic locus (2 mutants per million cells per generation). These results indicate that the mutation which rendered Y1 cells insensitive to ACTH likely was the same as that which led to forskolin-resistance. Furthermore, the mutation seemed to behave dominantly. Although the gene product altered by the mutation is unknown, it does not appear to be the catalytic subunit of the enzyme.
PURPOSE: Numerous orthopedic disorders involve periaxial rotation of long bones. We have developed and evaluated a computer-assisted method that segments a bone from X-ray CT, graphically unwraps the bone around its long axis into a surface plot (signature landscape), and measures periaxial rotation as the translation shift of the landscape. Bones in known rotations and partially segmented surfaces were used, respectively, to test accuracy and problematic situations in bone segmentation. METHOD: CT images of three chicken femora at known rotations were analyzed to determine their relative periaxial rotations, which were compared with the known rotations. RESULTS: The regression slope between measured and expected periaxial rotations was 1.005 +/- 0.003, with a maximum discrepancy of 2 degrees for same-bone and 3 degrees for interbone comparisons. Rasterization artifacts and the use of partial surfaces (with < 40% surface omission) resulted in a < 3 and < 1 degree error, respectively. CONCLUSION: The current method provides accurate and objective periaxial rotation measurements of a long bone.
Two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line fail to respond to ACTH with increased adenylyl cyclase activity and, as a consequence, are resistant to the steroidogenic effects of the hormone. As determined from Northern blot and RNase protection assays, ACTH resistance in these mutants results from the failure to accumulate ACTH receptor transcripts. The ACTH receptor gene appears to be present in these mutants as determined by Southern blot hybridization analysis and can be activated following the growth of the mutant cells as tumors in mice, suggesting that the ACTH receptor gene is modified in a reversible manner. When mutant cells are transformed with a gene encoding the mouse beta 2-adrenergic receptor they respond to beta-adrenergic agonists with increased adenylyl cyclase activity in a manner that is indistinguishable from a similarly transformed parent Y1 cell line. These results suggest that the adenylyl cyclase system in the mutants is otherwise intact and that the failure to express ACTH receptor transcripts limits the responsiveness of these clones to the hormone.
Subtilisin, a bacterial serine protease, is secreted as pre-pro-subtilisin. Previously, we demonstrated that the pro-peptide moiety of intact pro-subtilisin can guide the folding of inactive protein to active enzyme both in an intramolecular (6) and intermolecular manner (18). Herein is reported the total chemical synthesis of the pro-sequence (77 amino acids) of pre-pro-subtilisin BPN' carried out by solid phase methods. The structure was confirmed by both sequencing and amino acid analysis of the fragment peptides resulting from a V-8 protease digest. Preliminary studies indicate that the synthetic pro-peptide itself can renature denatured subtilisin BPN'. This study demonstrates a novel method for examining protein folding with the aid of exogenously added synthetic peptides.