Correlation of open cell-attached and excised patch clamp techniques

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na⁺ channel, located on the apical surface of A₆ cells, to determine whether the nystatininduced open cell-attached patch could serve as an alternative configuration.Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and E _rev was zero in symmetrical Na⁺ solutions, indicating similar Na⁺ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3–4pS) and E _rev (75 mV), when the bath was perfused with a high K:low Na solution (E _Na=80 mV), were nearly equal in both patch configurations.Our results therefore indicate that the nystatininduced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.We thank Dr. Olaf S. Andersen for his suggestions in the development of the open cell-attached recording technique. This work was supported by a National Institutes of Health grant (DK-18061)     

Long-term follow-up of patients with myocarditis and idiopathic dilated cardiomyopathy after immunomodulatory therapy

Abstract The randomized clinical trial with interferon-α (IFN) or thymic hormones versus conventional therapy was conducted in patients with myocarditis and idiopathic dilated cardiomyopathy (IDC). We enrolled 180 patients to receive IFN (3–5 million units per day) for 3 months, thymomodulin (10 mg three times per week) for 2 months, or conventional therapy alone. Patients were followed-up for 7 years after the end of treatment. Left ventricular function, exercise tolerance and survival rate were significantly better at long-term follow-up in patients treated with IFN or thymomodulin, than in conventionally treated patients. These results implicate that immune modulating therapy might represent important contribution in the treatment of myocarditis and IDC.     

Action of low-density lipoprotein and compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, on the synthesis of dolichol-linked oligosaccharides and low-density-lipoprotein receptor in human skin fibroblasts.

To explore whether there is an inter-relationship between the rate of low-density (LD) lipoprotein binding to its receptor and the formation of dolichol-linked oligosaccharides, experiments were performed with human fibroblasts where the synthesis of lipoprotein receptor and dolichyl saccharides was under control of LD lipoprotein and compactin. Pretreatment of the cells with nonlabelled LD lipoprotein resulted in a suppression of both the binding of 125I-labelled LD lipoprotein to the receptor and the synthesis of dolichyl saccharides from [14C]acetate and [3H]mannose, but not from [3H]mevalonolactone. Compactin, in contrast, inhibited only the formation of dolichol-linked oligosaccharides. Mevalonolactone (1 microM) abolished the inhibitory effect of LD lipoprotein on dolichyl saccharide formation, but was not able to restore the receptor-binding capacity, thus suggesting that the synthesis of lipoprotein receptor is not coupled to the formation of dolichyl saccharides.     

Protein dynamics. Comparative investigation on heme-proteins with different physiological roles.

We report the low temperature carbon monoxide recombination kinetics after photolysis and the temperature dependence of the visible absorption spectra of the isolated alpha SH-CO and beta SH-CO subunits from human hemoglobin A in ethylene glycol/water and in glycerol/water mixtures. Kinetic measurements on sperm whale (Physeter catodon) myoglobin and previously published optical spectroscopy data on the latter protein and on human hemoglobin A, in both solvents, (Cordone, L., A. Cupane, M. Leone, E. Vitrano, and D. Bulone. 1988. J. Mol. Biol. 199:312-218) are taken as reference. Low temperature flash photolysis data are analyzed within the multiple substates model proposed by Frauenfelder and co-workers (Austin, R. H., K. W. Beeson, L. Eisenstein, H. Frauenfelder, and I. C. Gunsalus. 1975. Biochemistry. 14:5355-5373). Within this model a distribution of activation enthalpies for ligand binding accounts for the structural heterogeneity of the protein, while the preexponential factor, containing also the entropic contribution to the free energy of the process, is considered to be constant for all conformational substates. Optical spectra are deconvoluted in gaussian components and the temperature dependence of the moments of the resulting bands is analyzed, within the harmonic Frank-Condon approximation, to obtain information on the stereodynamic properties of the heme pocket. The kinetic and spectral parameters thus obtained are found to be protein dependent also with respect to their sensitivity to changes in the composition of the external medium. A close correlation between the kinetic and spectral features is observed for the proteins examined under all experimental conditions studied. The results reported are discussed in terms of differences in the heme pocket structure and in the conformational heterogeneity among the various proteins, as related to their different capability to accommodate constraints imposed by the external medium.     

Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.