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R K Johnson

Publications and source records attributed to R K Johnson.

At least 127 records · Page 7Linked to original sources
PubMed1992

Antagonism of TGF-alpha receptor binding and TGF-alpha induced stimulation of cell proliferation by methyl pheophorbides.

Over 4,500 natural product extracts were screened for their abilities to inhibit binding of radiolabeled TGF-alpha to A431 cells; several plant extracts were identified as potential leads with IC50 values of less than 30 micrograms/mL. The active components of one extract were purified to homogeneity and identified as the porphyrin structures, methyl pheophorbides a and b. These compounds inhibited both TGF-alpha receptor binding and the TGF-alpha induced proliferation of NRK-49F cells in soft agar. To construct a structure-function relationship, a series of commercially available porphyrin derivatives was evaluated. The most potent compound, hematoporphyrin IX, inhibited TGF-alpha functions in a dose-dependent fashion with IC50 values slightly lower than the methyl pheophorbides. Further studies revealed that inhibition of TGF-alpha binding was light dependent and that inhibition did not involve direct competition of porphyrins for the TGF-alpha binding site. To determine the specificity of inhibition, the porphyrins were tested in a number of other receptor-ligand assays. TNF-alpha and beta-adrenoceptor bindings were unaffected, whereas IL-1 beta binding to EL-4 membranes and platelet-derived growth factor induced thymidine incorporation in NIH-3T3 cells were both antagonized by the most active porphyrins. Inhibition of TGF-beta binding to NRK-49F cells and TGF-beta-induced growth of AKR-2B cells was also observed. In summary, we report that methyl pheophorbides are naturally occurring, photodynamic antagonists of TGF-alpha, and although the inhibitory properties of these molecules were not confined to TGF-alpha alone, some level of receptor selectivity was observed.

Animals↗
PubMed1992

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.

Animals↗
PubMed1992

Dietary and anthropometric assessment of adults with cerebral palsy.

Research on nutrition and cerebral palsy (CP) has been directed at newborns and young children, leaving practitioners with a limited understanding of the nutritional status of the adult population. The purpose of this study was to determine the anthropometric profile and nutrient intakes of 86 adults with cerebral palsy. When compared with growth standards for healthy individuals, the mean body fat percentages and body mass indexes of both men and women with CP were within the normal range. However, 40% of the sample had heights below the 5th percentile for their age and gender, indicating permanent growth stunting. In general, the diets of these individuals were similar to the typical American diet. Both men and women had low nutrient adequacy ratios (NARs) for calcium (0.86 and 0.76, respectively); the women also had low NARs for iron (0.69) and niacin (0.86). Although nutrients obtained from supplements were not included in the NAR score, 26% of the men and 50% of the women reported using nutritional supplements. Fifty-five percent of the sample reported feeding problems. Multivariate analysis illustrated that individuals who participated in regular exercise programs had significantly higher mean adequacy ratios and lower body fat percentages than those who did not exercise regularly.

Adipose Tissue↗
PubMed1992

Maternal employment and the quality of young children's diets: empirical evidence based on the 1987-1988 Nationwide Food Consumption Survey.

Data from 442 children, aged 2 to 5, who were participants in the 1987-1988 Nationwide Food Consumption Survey, were examined to determine the effect of maternal employment on the quality of their diets. Diet quality was assessed by examining nutrient adequacy and nutrient overconsumption using the 3-day average of one 24-hour recall and 2 days of written diet records. To assess dietary adequacy, a mean adequacy ratio of the four nutrients (zinc, vitamin E, iron, and calcium) for which 30% or more of the children fell below 77% of the Recommended Dietary Allowances was constructed. Percent calories from fat and saturated fat, and intake of cholesterol and sodium were examined to assess overconsumption. Multiple correlation regression analysis was used to control for household income, maternal education and age, child's age, race, number of siblings aged 5 or younger, presence of male head of household, and number of meals eaten away from home. Maternal employment did not contribute significantly to the variation in any of the dietary variables. Although a number of dietary problems existed among the sample children, the variation in intakes of these nutrients was not directly related to maternal employment status.

Analysis of Variance↗
PubMed1992

Stabilization of the distal ulna by transfer of the pronator quadratus origin.

Surgical and nonsurgical treatment of distal radioulnar joint instability has characteristically been unsatisfactory. This present paper presents a surgical method involving the removal and transfer of the origin of the pronator quadratus to a more dorsal position, which effectively gives a dynamic stabilization to the joint. Temporary internal immobilization is performed with a transverse Kirschner wire that is removed at four weeks, and graded physical therapy is started at that time.

Bone Wires↗
PubMed1991

The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase with an N-terminal signal sequence.

Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene [Haendler et al., Gene 83 (1989) 39-46]. We recently identified a second Cyp-related gene in yeast, CYP2 [Koser et al., Nucleic Acids Res. 18 (1990) 1643] which predicts a protein with a hydrophobic leader sequence. A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli. The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA. The CYP2 gene is genetically unlinked to CYP1. As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability. Disruption of all three of the known yeast PPIase-encoding genes [CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence. Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity. This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast. Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.

Amino Acid Isomerases↗
PubMed1991

Synergistic cell killing by ionizing radiation and topoisomerase I inhibitor topotecan (SK&F 104864).

Topotecan (SK&F 104864), a water-soluble analogue of the topoisomerase I inhibitor camptothecin, is currently in Phase II clinical trial for solid tumors. We have characterized topotecan in terms of its effect upon gamma-radiation-induced cell killing. In colony formation experiments, subtoxic concentrations of topotecan (2 microM) potentiated radiation-induced killing of exponentially growing Chinese hamster ovary or P388 murine leukemia cultured cells. Survival curve shoulders were reduced; the slopes of the exponential portions of the curves were decreased to a small extent. D37 and D10 (radiation dose resulting in 37 and 10% survival of colony-forming ability) values were reduced by approximately 60 and 50%, respectively, in the case of Chinese hamster ovary cells. In P388 cells, topotecan reduced D37 by 35 to 40% and D10 by 20 to 25%. Potentiation of radiation-induced cell killing by topotecan was absolutely dependent upon the presence of the topoisomerase I inhibitor during the first few (less than 30) min after irradiation. Association of topoisomerase I with this effect was confirmed in studies of Chinese hamster ovary cells previously made resistant to camptothecin (and cross-resistant to topotecan), resulting in decreased cellular content of topoisomerase I. These cells were found to be 2- to 3-fold hypersensitive to gamma-radiation-induced killing. P388 camptothecin-resistant cells were further sensitized to the lethal effects of ionizing radiation by nontoxic treatment with the topoisomerase II inhibitor novobiocin, consistent with increased dependence of topoisomerase I-deficient cells upon topoisomerase II.

Animals↗
PubMed1991

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Animals↗
PubMed1991

Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Amino Acid Isomerases↗
PubMed1991

Estimates of genetic parameters for direct and maternal effects on embryonic survival in swine.

Survival of 16,838 potential embryos was determined by counting corpora lutea and fetuses at 50 d of gestation for 1,081 litters by 225 sires. These data, coded as 1 or 0 depending on whether an ovulation was represented by a fetus, were used to estimate direct and maternal additive genetic variances and their covariance for embryonic survival. Data were from first-parity gilts of a Large White-Landrace composite population subdivided into two lines, one selected for an index of ovulation rate and embryonic survival for seven generations and a contemporary control line. Variance components were obtained by ANOVA and expectations of covariances among relatives and by derivative-free restricted maximum likelihood (DFREML) in an animal model. As a trait of the embryo, heritability of direct effects obtained with ANOVA was 3.8%, heritability of maternal effects was 1.5%, and the genetic correlation between them was -.51. After adjustment of embryonic survival for ovulation rate, lower estimates of each parameter were obtained with ANOVA. Heritability of embryonic survival as a trait of the dam was 9 to 10%. Estimates of heritability of both direct and maternal effects obtained with DFREML were less than 1% and the genetic correlation between them was -.64. When survival of embryos from only those dams with 15 or more ovulations was analyzed, heritability of maternal effects was 4.4%. Estimates of common environmental effects on embryonic survival ranged from 5 to 7%.

Analysis of Variance↗
PubMed1991

Comparison of methods of estimating variance components in pigs.

Components of variance due to average effects of genes (sigma 2g), environmental effects common to littermates (sigma 2c), and environmental effects peculiar to individual pigs (sigma 2e) were estimated (--) by the Pseudo Expectation Approach (PE). Data were litter size (LS), backfat (BF; centimeter) and ADG (kilograms/day) collected from the Nebraska Gene Pool swine population between 1967 and 1986. Mean square errors (MSE) for h--2 and c--2 (sigma--2g and sigma--2c divided by phenotypic variance) by PE and nested ANOVA and h2 estimated by offspring on parent regression (REGOP) were evaluated using simulation of 200 repetitions of the Nebraska Gene Pool population. Parameter values for sigma 2g, sigma 2c, and sigma 2e used in simulations were PE estimates from the Gene Pool population. Estimates of h2 from PE were .18 +/- .06 for LS, .56 +/- .06 for BF, and .16 +/- .05 for ADG. Estimates of c2 from PE were .01 +/- .03 for LS, .09 +/- .02 for BF, and .19 +/- .03 for ADG. Compared with REGOP, PE yielded h--2 with smaller MSE for BF and ADG and larger MSE for LS. The MSE of PE was smaller than the MSE of the nested ANOVA estimate for all estimates and traits. These results were interpreted to suggest that considerable gains in precision in estimation of genetic parameters could be achieved by accounting for all relationships in lieu of accounting for only half- and full-sib relationships or parent-offspring relationships.

Adipose Tissue↗
PubMed1991

Effects of selection of data on estimates of breeding values by three methods for litter size, backfat, and average daily gain in swine.

Estimated breeding value was calculated based on individual phenotype (SP), an index of individual phenotype and full- and half-sib family averages (SI), or Best Linear Unbiased Prediction (BLUP). Traits considered were litter size (LS), backfat (BF), and ADG. Estimated breeding values were calculated using all data and after deletion of the poorest 5, 10, 15, or 20% of the records for BF and ADG, or 4.8, 8, 13, or 21% of the records for LS. When all data were used, expected genetic gain from BLUP was greater than for SP by 22, 7, and 31% and greater than for SI by 10, 4, and 21% for LS, BF, and ADG, respectively. Expected genetic gain was 4, 0, and 3% lower for LS, BF, and ADG, respectively, for selection on breeding values estimated by SI after the poorest 20% of the records were deleted compared with selection on estimates by SI using all the data. Genetic gain using BLUP on data with the poorest 20% of the records deleted was reduced by 5, 2, and 8% for LS, BF, and ADG, respectively, compared with genetic gain using BLUP on all the data. The advantage in genetic gain of BLUP, with 20% of the poorest records deleted, over SP was 15, 5, and 21% for LS, BF, and ADG, respectively. Although BLUP is affected to a greater degree by deletion of records than is SP or SI, selection of swine using BLUP on field data would improve response to selection over the use of SP or SI.

Adipose Tissue↗
PubMed1991

Direct responses to selection for increased litter size, decreased age at puberty, or random selection following selection for ovulation rate in swine.

Nine generations of selection for high ovulation rate were followed by two generations of random selection and then eight generations of selection for increased litter size at birth, decreased age at puberty, or continued random selection in the high ovulation rate line. A control line was maintained with random selection. Line means were regressed on generation number and on cumulative selection differentials to estimate responses to selection and realized heritabilities. Genetic parameters also were estimated by mixed-model procedures, and genetic trends were estimated with an animal model. Response to selection for ovulation rate was about 3.7 eggs. Response in litter size to selection for ovulation rate was .089 +/- .058 pigs per generation. Average differences between the high ovulation rate and control lines over generations 10 to 20 were 2.86 corpora lutea and .74 pigs (P less than .05). The regression estimate of total response to selection for litter size was 1.06 pigs per litter (P less than .01), and the realized heritability was .15 +/- .05. When the animal model was used, the estimate of response was .48 pigs per litter. Total response in litter size to selection for ovulation rate and then litter size was estimated to be 1.8 and 1.4 pigs by the two methods. Total response to selection for decreased age at puberty was estimated to be -15.7 d (P less than .01) when data were analyzed by regression (realized heritability of .25 +/- .05) and -17.1 d using the animal model. No changes in litter size occurred in the line selected for decreased age at puberty. Analyses by regression methods and mixed-model procedures gave similar estimates of responses and very similar estimates of heritabilities.

Age Factors↗
PubMed1991

Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.

Animals↗
PubMed1991

Regulation of topoisomerase I and II activities by cyclic nucleotide- and phospholipid-dependent protein kinases. Effects of interactions between the two transduction pathways.

Incubation of cultured rat aortic smooth muscle cells (A-10) with activators of cyclic nucleotides resulted in transiently increased activity of extractable topoisomerase I or topoisomerase II. ANF, which induces cGMP accumulation, potentiated camptothecin-induced, topoisomerase I linked DNA strand breakage and increased the specific activity of extractable topoisomerase I (maximum activity 5-15 min after treatment), but had no effect on topoisomerase II activity. These effects are similar to those reported for AVP and phorbol esters, activators of protein kinase C. Forskolin and isoproterenol, which induce cAMP accumulation, activated extractable topoisomerase II (maximum 5-15 min after treatment), but not topoisomerase I. Permeable cyclic nucleotide analogs dBcAMP and 8BrcGMP selectively activated extractable topoisomerase II and topoisomerase I activities, respectively. Activation of topoisomerase I by either AVP or PdBu was attenuated by cotreatment with 8BrcGMP or dBcAMP, and activation of topoisomerase II by dBcAMP was attenuated by cotreatment with AVP or PdBU, suggesting that elements of the protein kinase C and the cyclic nucleotide linked signal-transduction pathways can interact to modify nuclear enzymic activity. IBMX, which elevates intracellular cAMP and cGMP, increased the extractable activities of both topoisomerase I and topoisomerase II. Thus, topoisomerase activity in cells may be governed in part by cyclic nucleotide levels.

Animals↗
PubMed1991

Expression of human recombinant cAMP phosphodiesterase isozyme IV reverses growth arrest phenotypes in phosphodiesterase-deficient yeast.

The low-Km cAMP-specific phosphodiesterases (PDEases) are of great pharmacological significance because of their involvement in regulating cAMP concentrations, which, in turn, are responsible for mediating the cellular response to extracellular signals such as hormones and neurotransmitters. We recently reported the isolation of a cDNA clone that encodes a human monocyte low-Km, rolipram-sensitive, cAMP PDEase (isozyme IV). We have engineered the inducible expression of this human PDEase in yeast. Cells of Saccharomyces cerevisiae contain two genes that encode cAMP PDEases. PDEase-deficient mutants are viable but exhibit specific growth arrest phenotypes associated with elevated intracellular cAMP content; these phenotypes include heat shock sensitivity and the inability to grow on acetate as a carbon source. We show that functional expression of our human cAMP PDEase in a genetically engineered PDEase-deficient strain of S. cerevisiae reverses these aberrant phenotypes. Furthermore, under conditions for growth arrest, rolipram is cytotoxic to PDEase-deficient mutants expressing the human cAMP PDEase, indicating that it is capable of inhibiting the human recombinant enzyme in vivo. This system can be used in the development of a yeast cell-based assay for isozyme-selective inhibitors of the human recombinant cAMP PDEase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

In vivo development and in vitro characterization of a subclone of murine P388 leukemia resistant to bis(diphenylphosphine)ethane.

Bis(diphenylphosphine)ethane (DPPE) and its gold coordination complexes have demonstrated antitumor activity in transplantable tumor models. This report describes the development of a P388 cell line (P388/DPPEc) that is resistant to DPPE and its analogues and the in vitro characterization of the cross-resistance of this subline to various antitumor and cytotoxic agents. The P388/DPPE tumor cell line was developed by serial transplantation in DPPE-treated mice. Resistance to DPPE was phenotypically stable. The P388/DPPE subline was cross-resistant to DPPE analogues and metal coordination complexes of DPPE. In addition, P388/DPPE cells were resistant to several mitochondrial uncouplers, including rhodamine-123, tetraphenylphosphonium, and carbonylcyanide-p-trifluro-methoxyphenyl hydrazone. P388/DPPE cells were less capable of sequestering and retaining 123Rh than were sensitive (P388/S) cells. Exposure to Au(DPPE)2+, a gold complex of DPPE with increased antitumor activity, resulted in a depletion of cellular ATP; the depletion was more rapid in the sensitive than the resistant cells. The rate of mitochondrial respiration, as measured by 14CO2 evolution from [6-14C]glucose, was greater in P388/S than in P388/DPPE. As with that evidenced for 123Rh, the cellular uptake of radiolabeled DPPE was decreased in P388/DPPEc cells. The results suggest that the basis for the resistance of this cell line may be an alteration in mitochondrial membrane potential. These data and the striking cross-resistance of P388/DPPE to mitochondrial uncouplers support the hypothesis that mitochondria may be one target involved in the cytotoxic or antitumor activities of these compounds. Mitochondria may also be causally related to the cytotoxic or antitumor activities, in that DPPE may be concentrated in cells via the presence of the inner mitochondrial membrane potential. Thus, P388/DPPE cells can serve as a tool to screen for and evaluate drugs that rely on affecting mitochondrial function, either mechanistically or causally, for their antitumor efficacy.

Adenosine Triphosphate↗