Ultranarrow conducting channels defined in GaAs-AlGaAs by low-energy ion damage

We have laterally patterned the narrowest conducting wires of two-dimensional electron gas (2DEG) material reported to date. The depletion induced by low-energy ion etching of GaAs-AlGaAs 2DEG structures was used to define narrow conducting channels. We employed high voltage electron beam lithography to create a range of channel geometries with widths as small as 75 nm. Using ion beam assisted etching by Cl2 gas and Ar ions with energies as low as 150 eV, conducting channels were defined by etching only through the thin GaAs cap layer. This slight etching is sufficient to entirely deplete the underlying material without necessitating exposure of the sidewalls that results in long lateral depletion lengths. At 4.2 K, without illumination, our narrowest wires retain a carrier density and mobility at least as high as that of the bulk 2DEG and exhibit quantized Hall effects. Aharonov–Bohm oscillations are seen in rings defined by this controlled etch-damage patterning. This patterning technique holds promise for creating one-dimensional conducting wires of even smaller sizes

cAMP-dependent Protein Kinase Activation Lowers Hepatocyte cAMP

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5\u27-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level

Discriminative Insulin Antagonism of Stimulatory Effects of Various cAMP Analogs on Adipocyte Lipolysis and Hepatocyte Glycogenolysis

Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity for the hepatocyte cAMP-dependent protein kinase isozymes and the low K(m), hormone-sensitive phosphodiesterases from both cell types. No correlation was found between insulin resistance of analog-stimulated hepatocyte phosphorylase and the concentration of analog required in vitro for half-maximal activation of either type I or type II cAMP-dependent protein kinase from hepatocytes. However, a good correlation was found between insulin resistance of cAMP analog-stimulated responses and the analog I50 values for the phosphodiesterase from both cell types. Using a new method capable of measuring hydrolysis at low analog concentrations, several of those analogs which had relatively low, but not high, phosphodiesterase I50 values were shown to be directly hydrolyzed by the low K(m) adipocyte phosphodiesterase. The insulin inhibition of cell responses when stimulated by hydrolyzable analogs, but not by poorly hydrolyzable analogs, is best explained by insulin stimulation of the low K(m) phosphodiesterases from both cell types

Two Classes of cAMP Analogs Which Are Selective for the Two Different cAMP-Binding Sites of Type II Protein Kinase Demonstrate Synergism When Added Together to Intact Adipocytes

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their K(a) for protein kinase and their lipophilicity rather than with either parameters alone. Some of the most potent lipolytic analogs had high I50 values for the particulate low K(m) cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations some analogs were as effective as optimal concentrations of epinephrine in stimulating glycerol release. The regulatory subunit of protein kinase has two different intrachain cAMP-binding sites and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1 and analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2 (Rannels, S.R., and Corbin, J.D. (1980) J. Biol. Chem. 255, 7085-7088). Thus, C-8 and C-6 analogs were tested in combination to stimulate lipolysis in intact adipocytes and to activate protein kinase in vitro. Each process was stimulated synergistically by a combination of a C-6 and C-8 analog. Two C-8 analogs or two C-6 analogs added together did not cause synergism of either process. For both lipolysis and protein kinase activation, C-8 thio analogs acted more synergistically than C-8 amino analogs when incubated in combination with C-6 analogs, a characteristic of type II protein kinase. It is concluded that the observed synergism of lipolysis is due to binding of cAMP analogs to both intrachain sites and that it is the type II protein kinase isozyme which is responsible for the lipolytic response

Microheterogeneity of Type II cAMP-Dependent Protein Kinase in Various Mammalian Species and Tissues

Excluding autophosphorylated species, at least six forms of the regulatory subunit of type II cAMP-dependent protein kinase (R(II)) from various mammalian tissues were identified by sodium dodecyl sulfate (SDS) gel electrophoresis of purified samples and of crude preparations photoaffinity labeled with 8-azido[32P] cAMP and by gel filtration. After autophosphorylation some heart R(II) forms termed type IIA (bovine, porcine, equine, and dog) shifted to a more slowly migrating band on SDS gels while others termed type IIB (rat, guinea pig, rabbit, and monkey) did not detectably shift. Both subclasses of R(II) exhibited variation in apparent M(r) on SDS gels. Bovine and porcine heart nonautophosphorylated R(II) had M(r) 56,000 and the autophosphorylated R(II) had M(r) 58,000, while dog and equine heart R(II) had M(r) 54,000 and 56,000 for these bands, respectively. Rat heart R(II) had M(r) 56,000 while rabbit and guinea pig heart R(II) had M(r) 52,000. More than one R(II) was found in different tissues of the same species. Rabbit skeletal muscle contained a M(r) 56,000 IIB form. Bovine lung contained almost equal amounts of a IIA form apparently identical to that of bovine heart and a M(r) 52,000 IIB form similar to that which predominated in bovine brain. Rat adipose tissue, brain, and monkey heart contained predominantly a M(r) 51,000 IIB form. The rat liver M(r) 56,000 IIB form chromatographed differently from all other R(II) tested by gel filtration. Several lines of evidence indicated that the various forms of R(II) were not derived from one another through proteolysis or other processes. Each of the type II forms rapidly incorporated 0.3-1.0 mol of 32P per mol of subunit when incubated with [γ-32P]ATP and C subunit. Four of the forms tested were similar in the cAMP concentration dependence for activation of their corresponding holoenzymes and inhibited C subunit about equally. Each exhibited two components of [3H]cAMP dissociation, indicating two intrachain cAMP-binding sites, and the dissociation rates for the respective sites, and the dissociation rates for the respective sites were similar

Short-Term Feedback Regulation of cAMP by Accelerated Degradation in Rat Tissues

A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes. The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pClΦS-cAMP (where 8-pClΦS- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were unoccupied at concentrations of 8-pClΦS-cAMP that produced maximal cAMP lowering. Thus, the possibility that 8-pClΦS-cAMP lowered cAMP by displacing it from protein kinase binding sites, making it available for hydrolysis, seemed unlikely. In adipocytes, the lowering of cAMP by 8-pClΦS-cAMP occurred in parallel with increases in lipolysis and activation of low K(m) phosphodiesterase, suggesting that the phosphodiesterase was responsible for the cAMP lowering. Further evidence for this assertion was the finding that in hepatocytes preloaded with low concentrations of 8-pClΦS-cAMP, glucagon lowered 8-pClΦS-cAMP by about 50%, an amount similar to the cAMP lowering observed with 8-pClΦS-cAMP treatment. The results were consistent with a cAMP-dependent protein kinase-catalyzed activation of a phosphodiesterase and suggested that 8-pClΦS-cAMP-mediated hydrolysis of cAMP mimicked a physiologically significant response. The observation of this phenomenon in several tissues further suggested that it may a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of cAMP

Assessing binary mixture effects from genotoxic and endocrine disrupting environmental contaminants using infrared spectroscopy

Benzo[a]pyrene (B[a]P), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent contaminants and concern has arisen over co-exposure of organisms when the chemicals exist in mixtures. Herein, attenuated total reflection Fouriertransform infrared (ATR-FTIR) spectroscopy was used to identify biochemical alterations induced in cells by single and binary mixtures of these environmental chemicals. It was also investigated as a method to identify if interactions are occurring in mixtures and as a possible tool to predict mixture effects. Mallard fibroblasts were treated with single and binary mixtures of B[a]P, PCB126, PCB153, BDE47 and BDE209. Comparison of observed spectra from cells treated with binary mixtures with expected additive spectra, which were created from individual exposure spectra, indicated that in many areas of the spectrum, less-than-additive binary mixture effects may occur. However, possible greater-than-additive alterations were identified in the 1650-1750 cm-1 lipid region and may demonstrate a common mechanism of B[a]P and PCBs or PBDEs, which can enhance toxicity in mixtures

Folding of a donor–acceptor polyrotaxane by using noncovalent bonding interactions

Mechanically interlocked compounds, such as bistable catenanes and bistable rotaxanes, have been used to bring about actuation in nanoelectromechanical systems (NEMS) and molecular electronic devices (MEDs). The elaboration of the structural features of such rotaxanes into macromolecular materials might allow the utilization of molecular motion to impact their bulk properties. We report here the synthesis and characterization of polymers that contain π electron-donating 1,5-dioxynaphthalene (DNP) units encircled by cyclobis(paraquat-p-phenylene) (CBPQT4+), a π electron-accepting tetracationic cyclophane, synthesized by using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The polyrotaxanes adopt a well defined “folded” secondary structure by virtue of the judicious design of two DNP-containing monomers with different binding affinities for CBPQT4+. This efficient approach to the preparation of polyrotaxanes, taken alongside the initial investigations of their chemical properties, sets the stage for the preparation of a previously undescribed class of macromolecular architectures