Mechanisms of use-dependent block of sodium channels in excitable membranes by local anesthetics

Many local anesthetics promote reduction in sodium current during repetitive stimulation of excitable membranes. Use-, frequency-, and voltage-dependent responses describe patterns of peak INa when pulse width, pulse frequency, and pulse amplitude are varied. Such responses can be viewed as reflecting voltage-sensitive shifts in equilibrium between conducting, unblocked channels and nonconducting, blocked channels. The modulated-receptor hypothesis postulates shifts in equilibrium as the result of a variable-affinity receptor and modified inactivation gate kinetics in drug-complexed channels. An alternative view considers drug blocking in the absence of these two features. We propose that drug binds to a constant-affinity channel receptor where receptor access is regulated by the channel gates. Specifically, we view channel binding sites as guarded by the channel gate conformation, so that unlike receptors where ligands have continuous access, blocking agent access is variable during the course of an action potential. During the course of an action potential, the m and h gates change conformation in response to transmembrane potential. Conducting channels with both gates open leave the binding site unguarded and thus accessible to drug, whereas nonconducting channels, with gates in the closed conformation, act to restrict drug access to unbound receptors and possibly to trap drug in drug-complexed channels. We develop analytical expressions characterizing guarded receptors as "apparently" variable-affinity binding sites and predicting shifts in "apparent" channel inactivation in the hyperpolarizing direction. These results were confirmed with computer simulations. Furthermore, these results are in quantitative agreement with recent investigations of lidocaine binding in cardiac sodium channels.     

Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?

Summary The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tc^r region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.     

Biosynthesis of apolipoprotein C-III in rat liver and small intestinal mucosa

We have examined the biosynthesis of rat apolipoprotein C-III in the small intestine and liver. The primary translation product of its mRNA was recovered from wheat germ and ascites cell-free systems. Comparison of its NH2-terminal sequence with the NH2 terminus of plasma high density lipoprotein-associated apolipoprotein C-III showed that apo-C-III was initially synthesized as a preprotein with a 20 amino acid long NH2-terminal extension: Met-X-X-X-Met-Leu-Leu-X-X-Ala-Leu-X-Ala-Leu-Leu-Ala-X-Ala-X-Ala. Co-translational cleavage of the cell-free translation product by signal peptidase generated a polypeptide with the same NH2 terminus as the mature protein (X-Glu-X-Glu-Gly-Ser-Leu-Leu-Leu-Gly-Ser-Met). Therefore, this apolipoprotein does not undergo post-translational proteolytic processing like two other high density lipoprotein-affiliated proteins, proapo-A-I and proapo-A-II. The mRNA encoding apolipoprotein C-III comprises 0.4% of the translatable RNA species in adult rat liver and 0.14% of the translatable RNA species in small intestinal epithelium. Acute fat feeding with a triglyceride meal resulted in a 2-fold increase in intestinal preapo-C-III mRNA accumulation but no change in the levels of preproapo-A-I mRNA. Thus, the acute response of the apo-A-I and C-III genes to triacylglycerol absorption differs.     

Steps in the stabilization of newly packaged DNA during phage P22 morphogenesis

The protein products of three adjacent P22 genes, 4, 10 and 26, are required for the stabilization of DNA newly packaged into P22 phage capsids. We have isolated unstable DNA containing capsids from cells infected with mutants defective in these genes. All three classes could be converted into mature phage in vitro, confirming that they represent intermediates in particle maturation. The first of the three proteins to add to the newly filled capsids is gp4, followed by gp10 and gp26. The active form of gp4 sediments at 3 S, while the active forms of both gp10 and gp26 sediment at 5 S. These soluble subunits appear to polymerize onto the newly filled capsids to form the neck of the mature phage, the channel for DNA injection. Since gp4 is the first protein to act after DNA packaging, the unstable DNA containing capsids from 4- -infected cells must represent the direct product of the packaging of DNA into procapsids. The major fraction of these capsids lost activity with a half-life of 1.1 minutes at 23 degrees C, though they were much more stable at 0 degree C. Electron microscopic observations indicated that the loss of activity was due to the DNA exiting from the incomplete capsids. The marginal stability of the condensed DNA molecules within capsids is consistent with models of ATP-driven condensation and spontaneous DNA ejection. The basis of the stability of these highly condensed molecules remains to be determined.     

Proteolytic processing and compartmentalization of the primary translation products of mammalian apolipoprotein mRNAs

The steps involved in the initial assembly of apolipoproteins and lipids into supramolecular arrays (nascent lipoprotein particles) are largely unknown. Examination of the proteolytic processing and compartmentalization of the primary translation products of apolipoprotein mRNAs represents one approach to deciphering the molecular details of lipoprotein assembly. The structures of the primary translation products of seven mammalian apolipoprotein mRNAs has been determined in the past several years. The organization of apolipoprotein signal peptides is typical of eukaryotic prepeptides, although an unusual degree of sequence conservation is present among the signal segments of apo AI, AIV, and E. For those apolipoprotein sequences studied in detail, SRP-dependent cotranslational translocation and proteolytic processing appears to be highly efficient and results in sequestration of the processed protein within the lumen of the endoplasmic reticulum (ER). However the mechanism by which these lipid-binding proteins avoid arrest during their translocation through the lipid bilayer of the ER membrane remains obscure. The two principal human HDL apolipoproteins undergo novel extracellular post-translational proteolytic processing, which results in removal of nonhomologous propeptides. The proteases responsible for proapo AI and AII processing appear to be different. The processing of these proapolipoproteins provides a potential series of steps for regulating the ordered assembly of HDL constituents.     

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.     

Molecular heterogeneity of creatine kinase isoenzymes

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

Competitive adherence as a mechanism of bacterial interference

To determine whether competition among bacteria for specific attachment sites on host cells can explain bacterial interference, Staphylococcus aureus strain 502A was tested in turn against two different nasal coryneforms, a strain of Pseudomonas aeruginosa, and a virulent strain of S. aureus, all in the presence of nasal mucosal cells. Particularly examined was the influence of sequence in which bacteria were presented to the nasal cells in comparison with initial mixtures and individual suspensions. Results paralleled those observed in clinical prophylaxis: the bacterium first to adhere to the epithelial cells was able, under uniform conditions, to interfere with the colonization of subsequently added bacteria. Secondary adherence was not eliminated but substantially reduced, and was probably related to steric blockage by the initial colonizer and its particular ability to dissociate from the host cell.     

Characterization of an Infectivity Assay for the Ribonucleic Acid of Bacteriophage MS2

An infectivity assay for MS2 ribonucleic acid (RNA), which uses bacterial spheroplasts of an F(-) strain of Escherichia coli, has an efficiency of 10(-5) infected spheroplasts per RNA molecule. The characteristics of this assay and the influence of several parameters are presented. Important variables include the duration of exposure of the cells to lysozyme-ethylenediaminetetraacetic acid, the duration of exposure of the spheroplast stock to the RNA solutions before dilution, the concentration of RNA, and the presence of competing RNA. The growth kinetics of the virus in the infected spheroplasts and the extent of lysis have also been studied.     

Electrical Impedance of Isolated Amnion

The electrical impedance of the guinea pig amniotic membrane was measured, under standardized conditions, over the frequency range of 20 to 7000 cycles/second. This impedance can be represented analytically by a simple frequency-dependent function which is precisely of the form of the Debye relaxation equation. The observed data exhibit a broad dispersion centered at a frequency of 1050 cycles/second and a narrow distribution of time constants centered about 152 microseconds, both effects being due to the polydisperse nature of amniotic tissue. If the narrow time-constant distribution is approximated by a single time constant, amnion impedance can be simulated by a simple electrical circuit of frequency-independent elements. The Maxwell-Wagner interfacial treatment, although successfully adapted for cell suspensions, is shown to lose its quantitative significance in the case of the tightly structured amnion. In addition, determinations were made on the chemical composition of amniotic fluid, fetal blood and urine, and maternal blood and urine; the DC potential across the amniotic membrane was also measured.