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S Fetzer

Publications and source records attributed to S Fetzer.

13 recordsLinked to original sources
PubMed1997

The [U4/U6.U5] tri-snRNP-specific 27K protein is a novel SR protein that can be phosphorylated by the snRNP-associated protein kinase.

SR proteins play important roles in the recognition and selection of the 3' and 5' splice site of a given intron and contribute to the phosphorylation/dephosphorylation-mediated regulation of pre-mRNA splicing. Recent studies have demonstrated that the U1 snRNP is recruited to the 5' splice site by protein/protein interactions involving the SR domains of the U1-70K protein and SF2/ASF. Recently, it was suggested that SR proteins might also contribute to the binding of the [U4/U6.U5] tri-snRNP to the pre-spliceosome (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706), although it remains unclear whether these SR proteins interact with proteins of the tri-snRNP complex. As a first step toward the identification of proteins that could potentially mediate the integration of the [U4/U6.U5] tri-snRNP complex into the spliceosome, we investigated whether purified [U4/U6.U5] tri-snRNP complexes contain SR proteins. Three proteins in the tri-snRNP complex with approximate molecular weights of 27, 60, and 100 kDa were phosphorylated by purified snRNP-associated protein kinase, which has been shown previously to phosphorylate the serine/ arginine-rich domains of U1-70K and SF2/ASF (Woppmann A et al., 1993, Nucleic Acids Res 21:2815-2822). These proteins are thus prime candidates for novel tri-snRNP SR proteins. Here, we describe the biochemical and molecular characterization of the 27K protein. Analysis of a cDNA encoding the 27K protein revealed an N-terminal SR domain strongly homologous (54% identity) to the SR domain of the U1 snRNP-specific 70K protein. In contrast to many other SR proteins, the 27K protein does not contain an RNA-binding domain. The 27K protein can be phosphorylated in vitro by the snRNP-associated protein kinase and exhibits several isoelectric variants upon 2D gel electrophoresis. Thus, the tri-snRNP-specific 27K protein could potentially be involved in SR protein-mediated protein/protein interactions and, additionally, its phosphorylation state could modulate pre-mRNA splicing.

Amino Acid Sequence↗
PubMed1996

Low pH facilitates capsaicin responses in isolated sensory neurons of the rat.

The effects of capsaicin (CAPS; 30 nM, 300 nM, 3 microM) and acidic solutions (pH 6.6, 6.1, 5.6, 5.1) were studied in dorsal root ganglion (DRG) neurons from adult rats in short term culture using the whole cell patch-clamp technique and a system for fast drug application. At -60 mV holding potential, both CAPS 30 nM and 300 nM for 10 s did not induce a significant membrane current in pH 7.3. The first response to 3 microM CAPS at pH 7.3 yielded an inward current of 898 +/- 517 pA and with pH 6.1 the sustained proton-induced current was 365 +/- 153 pA. A more than additive current increase was observed when both agents were applied together even at subthreshold concentrations of CAPS or protons. Similar results were obtained at positive holding potential. Facilitation was also observed when extracellular pH 6.1 solution was applied immediately after discontinuation of 3 microM CAPS application but not when CAPS followed the application of pH 6.1 solution (n = 8). The proton-induced current as well as the CAPS-pH response both increased with proton concentration and showed the same short relaxation time relative to the CAPS response. The facilitation saturated near pH 5.6, and was present in repeated trials when responses to CAPS were markedly decreased due to tachyphylaxis. It is suggested that protonation of CAPS gated ion channels increases their open probability or conductance and modulates their kinetics.

Animals↗
PubMed1994

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes.

We have studied the regulation of intracellular pH (pHi), and HCO3(-)-dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pHi was 6.96 in both nominally CO2/HCO3(-)-free, HEPES-buffered saline (6.96 +/- 0.14; n = 48) and in a saline containing 5% CO2/24 mM HCO3- (6.96 +/- 0.18; n = 48) (at pH 7.4). Inhibition of the Na+/H+ exchange by amiloride (2 mM) caused a significant decrease of pHi in nominally CO2/HCO3(-)-free saline. Addition of CO2/HCO3- in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na+ also caused a fall of pHi, and addition of CO2/HCO3- in Na(+)-free saline evoked a further fall of pHi, while the outward current was reduced or even reversed. The stilbene 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM) reduced the pHi recovery from the CO2/HCO3(-)-evoked acidification, and blocked the prominent intracellular acidification upon removal of CO2/HCO3-. Removal of external Cl- had little effect on these pHi changes. Lowering the external pH from 7.4 to 6.6 in CO2/HCO3(-)-containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO2/HCO3-. The results suggest that the CO2/HCO3(-)-dependent current is partly due to a reversible bidirectional, electrogenic Na(+)-HCO3- cotransporter, which helps to regulate pHi in these cells. In addition, a prominent Na+/H+ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steady-state pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Influences of 7-alkyl substitution on the reversible binding of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene to DNA.

The effects of 7-alkyl substitution on the reversible intercalation of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BAD) to calf thymus DNA have been examined using time-resolved fluorescence spectroscopy. The results indicate that in 10(-3) M sodium cacodylate the binding constant of BAD is 1.8 x 10(3) M-1. 7-Ethyl substitution decreases the binding constant 1.6 times, while 7-methyl substitution increases the binding constant 1.7 times. UV Photoelectron data and results from ab initio molecular orbital calculations suggest that an increase in polarizability contributes to the increased binding accompanying methyl substitution. The decreased binding accompanying ethyl substitution arises from steric inhibition. The physical binding data correlates with the decrease in carcinogenic activity which occurs with 7-ethyl substitution of benz[a]anthracene metabolites.

Alkylation↗
PubMed1988

UV photoelectron spectroscopy and ab initio characterization of valence orbital structures and conformations of neutral phosphate esters.

The HeI UV photoelectron spectrum of trimethyl phosphate (TMP) has been measured and interpreted with the aid of SCF molecular orbital calculations carried out with STO-3G, STO-3G* and 4-31G basis functions. The photoelectron spectrum of TMP is more accurately reproduced by results from 4-31G calculations than by results from STO-3G or STO-3G* calculations. However, all three basis sets yield results which predict the same assignment of the photoelectron spectrum. Results at the 4-31G level indicate that whether calculations are based on crystallographic bond angles and bond lengths or on STO-3G optimized geometries has little effect on the energetic ordering of the upper occupied orbitals. The energetic ordering of orbitals is also found to be only weakly dependent upon the torsional angle phi, describing rotation of ester groups about P-O bonds and upon the torsional angle psi, describing rotation of methyl groups about C-O bonds. For trimethyl phosphate, with C3 symmetry, the vertical ionization potentials of the upper occupied orbitals are 10.81 eV (8e), 11.4 eV (9a), 11.93 eV (7e), 12.6-12.9 eV (8a and 6e), 14.4 eV (7a) and 15.0-16.0 eV (5e and 6a). Calculations at the 4-31G level indicate that many of the highest occupied orbitals in neutral dimethyl phosphate and methyl phosphate have energies and electron distributions similar to orbitals in TMP. For TMP, a search for optimized values of phi and psi has been carried out at the STO-3G*level. In agreement with previous NMR studies and with classical potential calculations, the STO-3G* results indicate that both the gauche (phi = 53.1 degrees) and anticlinal (phi = 141.9 degrees) conformations are thermally accessible. Also in agreement with the classical potential calculations, the STO-3G* results predict that in the all gauche conformation energy is minimized when the methyl groups assume a staggered geometry (psi = 60 degrees to 80 degrees) and that an energy maximum occurs for an eclipsed geometry (phi = 0 degrees to 20 degrees). A study of the dependence of optimized values of O-P-O ester bond angles on the torsional angles, phi, was carried out at the STO-3G, STO-3G* and 4-31G levels. The results demonstrate that for C3 symmetry, the coupling of O-P-O angles to phi is influence by repulsive steric interactions.

Chemical Phenomena↗