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S R Adams

Publications and source records attributed to S R Adams.

At least 37 records · Page 2Linked to original sources
PubMed1994

Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal.

The heat-stable inhibitor of cAMP-dependent protein kinase (PKI) was shown previously to export the kinase catalytic subunit (C) from the nucleus (Fantozzi, D. A., Harootunian, A. T., Wen, W., Taylor, S. S., Feramisco, J.R., Tsien, R. Y., and Meinkoth, J. L. (1994) J. Biol. Chem. 269, 2676-2686), in addition to its ability to inhibit kinase activity. In this study, the mechanism of PKI export is investigated. The injection of a C-PKI complex containing both labeled PKI and C-subunit revealed that both proteins exit the nucleus in unison. A fusion protein of C-subunit with glutathione S-transferase (GST) (140 kDa) cannot transverse the nuclear membrane in either direction, but can be exported from the nucleus when complexed with PKI, supporting the presence of a nuclear export signal (NES) in the C-PKI complex. Fusions of PKI alpha with GST (70 kDa) or PKI beta 1 with maltose-binding protein (MBP) (50 kDa) remain effective at exporting complexes with C-subunit. The export of C-PKI is also sensitive to temperature and energy depletion. Taken together, these results demonstrate that export is both energy- and temperature-dependent, but size-independent, consistent with an active signal-mediated export process. GST-PKI exits from the nucleus even in the absence of C-subunit, indicating that the NES resides entirely on PKI, but suggesting that fusion of PKI to GST leads to a conformational change that mimics the exposure of the NES caused by the binding of C. Since both PKI alpha and PKI beta 1 can export C-subunit, the predicted export signal is likely to reside on the residues conserved between PKI alpha and PKI beta 1.

Adenosine Triphosphate↗
PubMed1994

Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells.

We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.

Animals↗
PubMed1993

Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits were monitored by confocal fluorescence microscopy. Bath application of serotonin produced a much greater elevation of [cAMP] in the processes than in the central bodies of the neurons. The resulting gradients must drive a sizable centripetal flux of cAMP because direct microinjection of cAMP showed that it diffused readily. Perinuclear increases in [cAMP] slowly caused the translocation of the freed catalytic subunit into the nucleus to an extent proportional to the percentage of its dissociation from the regulatory subunit.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.

Animals↗
PubMed1993

Experimental infection of Anopheles gambiae s.s., Anopheles freeborni and Anopheles stephensi with Plasmodium malariae and Plasmodium brasilianum.

Susceptibility to infection of 2 strains of Anopheles gambiae s.s., An. freeborni and An. stephensi, was determined for 2 closely related malaria parasites, Plasmodium malariae and P. brasilianum. Neither strain of An. gambiae supported development of oocyst densities as great as the other 2 anopheline mosquitoes. The ZAN strain of An. gambiae s.s. from Zanzibar was more susceptible to infection with the strain of P. malariae from Uganda than the G-3 strain of An. gambiae s.s. from The Gambia. All species and strains of mosquitoes supported complete development to the presence of sporozoites in the salivary glands.

Animals↗
PubMed1992

Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging.

Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.

Adrenergic alpha-Antagonists↗
PubMed1991

Fluorescence ratio imaging of cyclic AMP in single cells.

Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.

Animals↗
PubMed1991

The initial use of a computer-controlled expert system in the treatment planning of Class II division 1 malocclusion.

A computer based expert system for providing orthodontic advice for Class II Division 1 malocclusion has been developed and evaluated. Treatment plans produced by the system for 31 non-prior approval cases drawn from material provided by the Dental Practice Board were compared with the treatments actually carried out by the practitioners. The shortcomings of the system are discussed.

Decision Trees↗
PubMed1990

Imaging and manipulation of cytosolic ions and messengers during cell activation.

Optical methods have recently become available for continuously imaging the free concentrations of important ions and second messengers such as calcium, sodium and hydrogen inside living cells. These ion levels are found to undergo remarkable changes upon stimulation of quiescent cells with growth factors known to stimulate phosphoinositide breakdown. In serum-starved REF-52 fibroblasts, growth factors such as serum, vasopressin, or PDGF (platelet-derived growth factor) cause intracellular [Na+] to increase from about 4 mM to 8 mM. If mitogen treatment is combined with pharmacological depolarization of the membrane potential, repetitive [Ca2+]i spikes result in these rat fibroblasts. The mechanism of this oscillation has been investigated by light-flash release of intracellular messengers such as inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), Ca2+, and diacylglycerol, as well as more traditional biochemical techniques. The key feedback pathway appears to be Ca2(+)-stimulation of phospholipase C production of Ins(1,4,5)P3.

Animals↗
PubMed1989

Evaluation of the possible direct effects of gonadotrophin-releasing hormone analogues on the monkey (Macaca mulatta) testis.

In Exp. 1, the effect of treatment with a GnRH agonist on basal concentrations of serum testosterone and peak values of serum testosterone after administration of hCG was determined. One group of adult male monkeys was treated with a low dose (5-10 micrograms/day) and a second group with a high dose (25 micrograms/day) of a GnRH agonist for 44 weeks. Basal and peak testosterone concentrations were both significantly reduced by GnRH agonist treatment in all groups compared to untreated control animals, but the % rise in serum testosterone above basal values in response to hCG administration was unchanged by agonist treatment. In Exp. 2, the GnRH agonist (100 or 400 ng) or a GnRH antagonist (4 micrograms) was infused into the testicular arteries of adult monkeys. The agonist did not alter testosterone concentrations in the testicular vein or testosterone and LH values in the femoral vein. In Exp. 3, testicular interstitial cells from monkeys were incubated with three concentrations (10(-9), 10(-7) and 10(-5)M) of the GnRH agonist or a GnRH antagonist with and without hCG. After 24 h, neither basal nor hCG-stimulated testosterone production was affected by the presence of the GnRH agonist or antagonist. The results from all 3 experiments clearly suggest that GnRH agonist treatment does not directly alter steroid production by the monkey testis.

Animals↗
PubMed1987

Effect of azotemia in dogs on the pharmacokinetics of pentamidine.

We used a new high-performance liquid-chromatography assay to study the pharmacokinetics of pentamidine isethionate given intravenously to 10 dogs before and after surgically induced renal failure. The presurgery peak serum concentration of pentamidine averaged 867 ng/ml and by 6 hr had fallen to 30 ng/ml. After surgery the peak and 6-hr concentrations were 780 ng/ml and 28 ng/ml. Mean total body clearance of pentamidine before surgery was 46.7 ml/min per kg, of which only 2.0 ml/min per kg was due to renal clearance had fallen 80%--to 0.4 ml/min per kg. Two dogs with azotemia and two control dogs received 14 daily infusions of pentamidine. Kinetic parameters measured after the last dose were not significantly different from those after the first dose, and the amount of pentamidine recovered from tissues was similar for dogs with azotemia and controls. In summary, because renal clearance of pentamidine accounted for such a small proportion of total body clearance, none of the parameters measured was affected significantly by moderate azotemia.

Amidines↗
PubMed1984

The arsonomethyl analogue of adenosine 5'-phosphate. An uncoupler of adenylate kinase.

Adenosine was converted into the arsonomethyl analogue of AMP. The reactions used provide a general route for converting an alcohol, R-CH2-OH, into the arsonomethyl analogue, R-CH2-CH2-AsO3H2, of its phosphate, R-CH2-O-PO3H2. The analogue of AMP proves to be a substrate for rabbit adenylate kinase, which shows a limiting velocity with it of 1/17 that with AMP, a Michaelis constant raised 70-fold to about 10 mM, and hence a specificity constant lowered about 1200-fold. The product of transfer of a phospho group from ATP to the analogue is, like all anhydrides of arsonic acids, unstable to hydrolysis, and so breaks down to yield orthophosphate and regenerate the analogue. Hence adenylate kinase is converted into an ATPase by the presence of the analogue.

Adenosine Monophosphate↗
PubMed1983

The arsonomethyl analogue of 3-phosphoglycerate.

4-Arsono-2-hydroxybutanoic acid, the analogue of 3-phosphoglycerate in which -CH2-AsO3H2 replaces -O-PO3H2, was synthesized. It proved to be a substrate for phosphoglycerate kinase. Its Michaelis constant was only slightly higher than that of the natural substrate, but its catalytic constant was about 1300 times smaller.

Arsenic↗
PubMed1977

Development of a guinea pig colony free of complement-fixing antibodies to parainfluenza virus.

Complement-fixing antibodies to parainfluenza 3 virus were found in Hartley strain [Cds: (HA)] guinea pigs from the authors' production colony. The prevalence and distribution of these antibodies were determined by testing guinea pigs of five age categories: 4 weeks, 8 weeks, 12 weeks, 6 months to 1 year, and over 1 year of age. Forty-seven percent (28 of 60) were positive to parainfluenza 3 antigen. Positive reactors were found in all age groups except those 8 weeks old. The 12-week-old group had the highest titers; the group over 1 year of age had the highest percentage of positives (92%). When 8-week-old guinea pigs were isolated, 55% were positive at some time between 8 and 34 weeks of age. The titers characteristically rose rapidly and then dropped slowly to low or undetectable levels. Four pairs of breeders over 6 months of age (most of whom were positive for parainfluenza 3 virus antibodies and, therefore, presumed to be immune to the virus) were isolated and allowed to breed. Their offspring were found to be free of complement-fixing antibodies to parainfluenza 3 virus.

Age Factors↗