Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin.

The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy     

Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

Determining the kinetics of membrane pores from patch clamp data without measuring the open and closed times

We present a new and very time saving way to determine kinetic rate constants from patch clamp data by using the correlation functions utilized in analyzing experiments with photon correlations.     

Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of water flow on transmembrane ionic currents in neurons of Helix pomatia and in squid giant axons

1. The effects of water flow through the membrane produced by an osmotic gradient on the ionic currents in Helix neurons and in squid giant axons were studied. 2. Outward water flow had a marked effect on the ionic currents. 3. Cell volume diminution in hypertonic solution was accompanied by a decrease in the number of functioning ionic channels in the neurons. 4. Decrease of the tonicity of the external 10(-8) M TTX-containing solution leads to a transient recovery of the action potentials of the squid.     

Identification and purification of overlapping cosmid clones of the region Xq24-qter of human X chromosome using HPLC.

The assembly of a large physical map of genomes requires simultaneous analysis of many cosmid clones for overlapping regions. The search for overlapping regions may be achieved by various means. High-performance liquid chromatography (HPLC) provides an alternative to gel electrophoresis since microgram amounts of each DNA fragment may be collected into individual test tubes for further analysis. HPLC has been used to identify overlapping cosmid clones from a pool of cosmid DNA containing the terminal portion of the long arm of the human X chromosome (Xq24-qter). Among 400 cosmids analyzed, 3 were shown to overlap.     

Transbilayer distribution of bromine in fluid bilayers containing a specifically brominated analogue of dioleoylphosphatidylcholine

We describe in this paper the transbilayer distribution of the bromines of the specifically halogenated phospholipid 1-oleoyl-2-(9,10-dibromostearoyl)-sn-glycero-3- phosphocholine (OBPC). The distribution was determined by X-ray diffraction of oriented multilayers of mixtures of OBPC and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 66% relative humidity by the general approach of Franks et al. (1978) [Nature 276, 530-532]. The bromine distribution of OBPC in the fluid L alpha phase is described accurately by a pair of Gaussian functions located 7.97 +/- 0.27 A from the center of the bilayer with l/e half-widths of 4.96 +/- 0.62 A. We find that OBPC bilayers are accurately described as DOPC bilayers with an additional bromine distribution centered at the position of the double bond of DOPC and conclude that OBPC is an excellent structural isomorph for DOPC under the conditions of these experiments. The distribution obtained is the complete and fully resolved transbilayer image of the halogen label because the broad distribution of the bromines is due entirely to thermal disorder and not to experimental limitations [Wiener, M. C., & White, S. H. (1991a) Biophys. J. 59, 162-173]. The observed width of the bromine distribution indicates that virtually all of the hydrocarbon interior is accessible to the bromines. The distance between the bromine/double-bond position and the headgroup phosphate position was determined from one-dimensional Patterson maps and found to be approximately 12 A. The application of accurately determined bromine distributions to the quantitative interpretation of fluorescence quenching experiments is discussed. A method for the self-consistent global analysis of diffraction data from mixtures that permits the use of data sets with different instrumental scale factors is developed in an Appendix.     

Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam: kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate

The kinetics of NADH consumption, oxygen uptake, and hydrogen peroxide production have been studied for norcamphor metabolism by cytochrome P-450cam. The kinetic deuterium isotope effects on these processes, with specifically deuteriated norcamphor, are 0.77, 1.22, and 1.16, respectively. Steady-state UV-visible spectroscopy indicates that transfer of the second electron to the dioxy ferrous P-450 is the rate-limiting step, as it is when camphor is the substrate. The inverse deuterium isotope effect for NADH consumption is consistent with an isotope-dependent branching between monooxygenase and oxidase activity, where these reactivities differ in their NADH:oxygen stoichiometries. However, no isotope-dependent redistribution of steady-state intermediates was detected by isotopic difference UV-visible spectroscopy in the presence of norcamphor. The kinetic isotope effects and steady-state spectral results suggest that the high-valent iron-oxo hydroxylating intermediate [FeO]3+ is reduced by NADH and the physiological electron-transfer proteins to afford water.