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Notes: . Signal transduction systems known to utilize G-proteins in higher eukaryotes undoubtedly evolved prior to the development of metazoa. Pharmacological evidence indicates that the ciliates Paramecium, Stentor, and Tetrahymena all utilize signaling systems similar to those found in mammals. However, there has been relatively little direct evidence for the existence of G-proteins in ciliates. Since highly conserved heterotrimeric G-proteins form the basis of receptor-coupled signal transduction systems in a wide variety of metazoa, it is of interest to know if these important signaling molecules were early to evolve and are present and functionally important in a wide variety of unicellular organisms. We have previously shown that mechanotransduction in Stentor is modulated by opiates in a manner that may involve pertussis toxin-sensitive G-proteins. Here we utilize drugs known to interact with G-proteins to further test for the involvement of these important signaling molecules in Stentor mechanotransduction. We present behavioral and electrophysiological data demonstrating that putative G-proteins in Stentor decrease mechanical sensitivity by modulating the mechanotransduction process. In addition, we report the partial cloning of 4 G-protein α-subunits from Stentor. We confirm that these clones are of Stentor origin and are transcribed. Furthermore, we employ antisense oligodeoxynucleotide-mediated knockout to demonstrate that these ciliate G-proteins exert a modulatory influence on Stentor behavior, and that a G1/G0-like clone mediates the inhibitory action of β-endorphin on mechanotransduction.

Notes: MyristoylCoA:protein N-myristoyltransferase (Nmt) catalyses the co-transiational, covalent attachment of myristate (C14:0) to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly-447 — Asp mutation was Introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37° C, as judged by in vitro kinetic studies of the wild-type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/Δnmt, and nmtΔ/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmtδ/nmtG447D strain required myristate for growth. This was true at both 24 and 37°C. Palmitate could not substitute for myristate. Incubation of nmtΔ/nmtG447D cells at 37° C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 μM myristate. Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/Δnmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmtΔ/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that Inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.

Notes: Summary In response to mechanical stimuli the protozoan,Stentor coeruleus, contracts in an all-or-none fashion and simultaneously reverses the direction of its ciliary beat. These behaviors have previously been shown to be correlated with the presence of a mechanoreceptor potential and all-or-none action potential (Wood 1970, 1973a). In the studies reported below the ionic bases of the resting, receptor and action potentials ofStentor were determined by use of intracellular microelectrodes penetrating animals chilled to 8.5–10 °C. The resting potential is most dependent on the extracellular concentration of KCl but some dependence on CaCl2 concentration was also observed. If allowance is made for the large increases in membrane conductance observed in solutions containing 2–8 mM KCl it is found that the resting potential data are well described by a modified form of the Goldman equation whereP Ca/P K = 0.068 andP Cl/P K = 0.072. The intracellular ionic activities (K i + = 13.1 mM, Cl i − = 9.9 mM, Ca i + = 0 mM) which provide the best fit of this equation to the resting potential data are in close agreement with the intracellular concentration values measured by flame microspectrophotometry (Ki=12.4 mM, Cli = 9.4 mM) except in the case of Cai where most of the intracellular concentration is presumed to be bound. 65 to 75 mV action potentials are produced by suprathreshold depolarizations but contractions were not generally seen in these chilled animals, only ciliary reversals. The action potential peak varies with CaCl2 concentration with a slope of 12.6 mV/10 fold change but varies only slightly with KCl or Cl− concentration. These peak potentials are well described by assuming that theP Ca/P k = 7.9 andP Cl/P K=1.0 at the time of the action potential peak. Depolarizing receptor potentials and brief inward receptor currents were observed for all forms of punctate and gross bodily mechanical stimulation employed. No evidence was found for any form of hyperpolarizing mechanoreceptor potentials as observed in some other ciliates. The reversal potential of the mechanoreceptor current varied with CaCl2 concentration in a manner similar to that of the action potential peak. As in the case of the action potential both theP Ca/P k andp cl/p k ratios appear to increase as a result of mechanical stimulation to 9.3–15 and 1.2–1.95 respectively. Mechanoreceptor currents are voltage dependent being increased when the membrane is depolarized above resting potential and decreased when the membrane is hyperpolarized. In general the electrophysiological characteristics ofStentor appear similar to those ofParamecium andStylonychia, but its resting membrane appears more selectively permeable to K+, it produces only depolarizing receptor potentials when mechanically stimulated and the initial action potential elicited by depolarizing current pulses can be all-or-none even in culture medium.

Notes: Summary Mechanoreceptor channels were localized by using the ligands, tubocurarine (TC), decamethonium (Deca), and gallamine (Gall), which have been shown to bind specifically to these channels. The binding of radioactively labeled TC (TC*) was found to be directly proportional to the cell surface area suggesting that the channels are uniformly distributed over the cell surface. Intracellular TC and Gall injections did not depress mechanical stimulus sensitivity though these drugs did depress sensitivity when applied extracellularly at the same concentrations; therefore, the ligand binding sites are on or near the external surface of the cell. Autoradiographs revealed that radioactively labeled Deca (Deca*) bound to the pigmented stripes but not to the ciliary stripes or membranellar band. Further,Stentor induced to shed their membranellar band through exposure to 8% urea were more sensitive to mechanical stimuli than were controls; therefore, the membranellar cilia do not appear to contain mechanoreceptor channels. Collectively, these data indicate that the mechanoreceptor channels are located in the somatic surface covering the pigmented stripes. The density of mechanoreceptor channels in the plasma membrane covering the somatic surface is tentatively estimated to be between 5500 and 14500 μm−2 based on the density of TC* binding, the apparent number of TC molecules binding per mechanoreceptor channel, and data suggesting that only one fifth to one fourth of the bound TC* is bound to structures in the plasma membrane.