Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP.

Olfactory receptor neurons respond to odorant stimulation with a rapid increase in intracellular cAMP that opens cyclic nucleotide-gated (cng) cation channels. cng channels in rat olfactory neurons are activated by cAMP in the low micromolar range and are outwardly rectifying. The cloned rat olfactory cng channel (rOCNC1), however, is much less sensitive to cAMP and exhibits very weak rectification. Here we describe the cloning and characterization of a second rat cng channel subunit, denoted rOCNC2. rOCNC2 does not form functional channels when expressed alone. When rOCNC1 and rOCNC2 are coexpressed, however, an outwardly rectifying cation conductance with cAMP sensitivity near that of the native channel is observed. In situ hybridization with probes specific for the two subunits shows that they are coexpressed in olfactory receptor neurons. These data indicate that the native olfactory cng channel is likely to be a heterooligomer of the rOCNC1 and rOCNC2 subunits.     

Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.     

Rapid application and removal of second messengers to cyclic nucleotide-gated channels from olfactory epithelium.

The last step in the second-messenger cascade mediating vertebrate olfactory transduction is the direct opening of a nonspecific cation channel by cAMP. The kinetic properties of this interaction are critical in determining the time course of the sensory response. To analyze these properties, excised inside-out membrane patches containing either the native channel from salamander olfactory-receptor neurons or a recombinant rat olfactory cyclic nucleotide-gated channel were exposed to short pulses of known concentrations of cAMP or cGMP to mimic a rapid and transient production of second messenger. Channel activity outlasted cyclic nucleotide pulses for several hundred milliseconds. This effect was due to an intrinsic property of the olfactory channel protein because it did not occur with cGMP-activated channels from retinal photoreceptors. Gating kinetics of the olfactory channel were both voltage and agonist dependent. These results demonstrate that the overall slow channel-gating kinetics could account for the difference in time course between the odor-induced changes in cAMP concentration and the subsequent sensory generator current.     

Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.     

The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.     

Rat sympathetic neurons and cardiac myocytes developing in microcultures: correlation of the fine structure of endings with neurotransmitter function in single neurons.

Microcultures containing single sympathetic principal neurons and small numbers of dissociated heart myocytes were prepared from newborn rats. After the transmitter properties of the neuron were studied by electrophysiological experiments, the microculture was examined with the electron microscope. Single neurons of either putative cholinergic or putative adrenergic character made morphological synapses on themselves (autapses), although only cholinargic autapses were detected electrophysiologically. Numerous axonal varicosities were present adjacent to the myocytes but no synaptic specializations were evident. After permanganate fixation to localize endogenous norepinephrine, the endings of neurons which appeared to secrete catecholamines contained many small granular vesicles, while the endings of neurons which appeared to secrete acetylcholine contained none. The endings of neurons which apparently secreted both catecholamines and acetylcholine contained only occasional small granular vesicles.     

Low Ba2+ and Ca2+ induce a sustained high probability of repolarization openings of L-type Ca2+ channels in hippocampal neurons: physiological implications.

Openings of single L-type Ca2+ channels following repolarization to negative membrane potentials from a depolarizing step (repolarization openings, ROs) have been described previously in brain cell preparations. However, these ROs have been reported to occur only infrequently. Here we report that the frequency of ROs in cell-attached patches of cultured rat hippocampal neurons can be increased dramatically by lowering the pipette Ba2+ concentration to 20 mM from the usual 90-110 mM. This increased opening probability can last for hundreds to thousands of milliseconds following repolarization. Current-voltage analyses of open probability show that the depolarization pulse threshold for inducing ROs in 20 mM Ba2+ is -10 to 0 mV but that the probability of ROs reaches maximal levels following depolarizing pulses that approach the apparent null (equilibrium) potential for Ba2+. Comparable current-voltage curves in 110 mM Ba2+ from a more positive holding potential (-50 mV) indicate that membrane surface charge screening accounts for some, but not all, of the effect of lowering the Ba2+ concentration. Consequently, current-dependent inactivation or some other ion-dependent mechanism (e.g., ion binding inside the pore) also appears to regulate this potentially major pathway of Ca2+ entry. A high probability of ROs also can be induced under relatively physiological conditions (5-ms depolarizing steps, 2-5 mM Ca2+ in the pipette). Thus, the high open probability state at negative potentials may underlie the long Ca2+ tail currents in hippocampus that were described previously and appears to have major implications for physiological functions (e.g., the slow Ca(2+)-dependent afterhyperpolarization), particularly in brain neurons.     

Factor VIII determination in patient's plasma and concentrates: a novel test equally suited for both matrices.

A novel assay for the determination of factor VIII (FVIII) is described. The assay uses a fluorescence-based detection system comparable with the common chromogenic test. At the same time, the assay is analogous to the clotting test as it does not involve pre-activation of FVIII. The assay was adjusted to perform equally well with patient's plasma and FVIII concentrates as samples. The combined employment of two different FVIII-deficient plasmas turned out to be of crucial importance in order to render the matrices as similar as possible to patient's plasma and, simultaneously, to obtain maximum sensitivity. Samples with a FVIII content down to 0.01 IU/ml are readily measured as are samples with a FVIII content of 1 IU/ml or somewhere in between. Upon dilution of samples, concentrates and plasma exhibited the same dose-response characteristics.     

Activation of lymphocytes by brominated nucleoside and cyclic nucleotide analogues: implications for the "second messenger" function of cyclic GMP.

The purine nucleoside guanosine, when derivatized at the C-8 position to give 8-bromoguanosine (8-BrGuo), acquires the capacity to stimulate high-level lymphocyte proliferation in the presence or absence of serum. Direct comparisons were undertaken to determine whether this activity is exerted only by virtue of the structural resemblance of 8-BrGuo to 8-bromo cyclic GMP (8-BrcGMP) (a known intracellular lymphocyte mitogen). They showed that, of the brominated guanosine derivatives studied, 8-BrGuo is the primary activator because (i) it is a far more potent lymphocyte activator than 8-BrcGMP, the order of mitogenic potency being 8-BrGuo greater than 8-bromo GMP (8-BrGMP) greater than 8-BrcGMP; (ii) it acts much more rapidly than 8-BrcGMP; (iii) it is not metabolized to 8-BrcGMP or cGMP; and (iv) it does not elevate intracellular cGMP content. cGMP is not likely to be the second messenger serving to activate B cells because (i) it does not induce significant proliferation unless brominated at the C-8 position; (ii) the brominated form is much less efficient than 8-BrGuo or 8-BrGMP; (iii) 8-BrGuo and many other mitogens do not increase intracellular cGMP; (iv) many agents that increase cGMP fail to initiate lymphocyte activation; (v) certain agents that increase cGMP (i.e., 15-hydroperoxyarachidonic acid, azide) inhibit lymphocyte activation; and (vi) addition of unbrominated cGMP to cultures stimulated with 8-BrGuo actually diminished stimulation. These data (i) indicate that, by interaction with cellular components, 8-BrGuo triggers high level lymphocyte activation and (ii) cast significant doubt on the role of cGMP as an intracellular second messenger in lymphocyte proliferation.     

Modulation of a transient K+ current in the pleural sensory neurons of Aplysia by serotonin and cAMP: implications for spike broadening.

To study the contribution of cAMP to the spike broadening produced by serotonin (5-HT) in the pleural sensory neurons of the tail withdrawal reflex, we utilized two phosphodiesterase-resistant cAMP analogs: the Sp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Sp-cAMP[S]), which activates protein kinase A, and the antagonist Rp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP[S]), agonist Sp-cAMP[S] was injected into the sensory neurons, it caused spike broadening comparable to that induced by 5-HT. In turn, the cAMP antagonist Rp-cAMP[S] blocked approximately 50% of the 5-HT-induced spike broadening. We next examined the K+ currents that are modulated by 5-HT and determined how these currents are affected by cAMP. Confirming Baxter and Byrne [(1989) J. Neurophysiol. 62, 665-679], we found that 5-HT modulated two currents, an S-type K+ current (IKS) as well as a transient and voltage-dependent K+ current (IKV). Rp-cAMP[S] blocked the reduction by 5-HT of the early phase of IKV in parallel with, and to the same degree (60%), as this inhibitor blocked the IKS and spike broadening. These results support the idea that in the pleural sensory neurons cAMP mediates a significant part of the spike broadening that accompanies short-term facilitation produced by 5-HT and that cAMP can produce spike broadening by modulating both IKV and IKS.     

Heteroatom-substituted fatty acid analogs as substrates for N-myristoyltransferase: an approach for studying both the enzymology and function of protein acylation.

Myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that transfers the myristoyl (14:0) moiety from myristoyl CoA thioester to nascent proteins, is remarkably specific for both peptide and fatty acyl CoA substrates. To investigate the interaction of NMT with fatty acyl CoA substrates, we have synthesized 10 oxygen- or sulfur-substituted fatty acid analogs. These analogs differ dramatically in hydrophobicity from naturally occurring fatty acids of similar length. As acylpeptides, sulfur-substituted myristic acid analogs migrate on reverse-phase HPLC like 11:0 or 12:0 fatty acids, while oxygen-substituted analogs migrate like 9:0 to 11:0 fatty acids. CoA thioesters of several of these analogs serve as good NMT substrates in vitro, implying that NMT selects fatty acyl substrates primarily on the basis of chain length rather than hydrophobicity. Myristelaidoyl (14:1, delta 9,10-trans) CoA is also a significantly better substrate than myristoleoyl (14:1, delta 9,10-cis) CoA. The fatty acyl group bound to NMT profoundly influences the rate of acylpeptide formation and the affinity of NMT for peptide substrates. However, the peptide substrate bound to NMT does not produce significant alterations in the enzyme's affinity for myristoyl CoA. In vitro characterization of these heteroatom substituted analogs suggests that they will be efficiently incorporated into proteins in vivo and may markedly alter acylprotein targeting and function.     

A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions

Glutamine/asparagine (Q/N)-rich domains have a high propensity to form self-propagating amyloid fibrils. This phenomenon underlies both prion-based inheritance in yeast and aggregation of a number of proteins involved in human neurodegenerative diseases. To examine the prevalence of this phenomenon, complete proteomic sequences of 31 organisms and several incomplete proteomic sequences were examined for Q/N-rich regions. We found that Q/N-rich regions are essentially absent from the thermophilic bacterial and archaeal proteomes. Moreover, the average Q/N content of the proteins in these organisms is markedly lower than in mesophilic bacteria and eukaryotes. Mesophilic bacterial proteomes contain a small number (0-4) of proteins with Q/N-rich regions. Remarkably, Q/N-rich domains are found in a much larger number of eukaryotic proteins (107-472 per proteome) with diverse biochemical functions. Analyses of these regions argue they have been evolutionarily selected perhaps as modular "polar zipper" protein-protein interaction domains. These data also provide a large pool of potential novel prion-forming proteins, two of which have recently been shown to behave as prions in yeast, thus suggesting that aggregation or prion-like regulation of protein function may be a normal regulatory process for many eukaryotic proteins with a wide variety of functions.     

Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis.

We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed.     

Spleen function in quartan malaria (due to Plasmodium inui): evidence for both protective and suppressive roles in host defense.

For investigation of the role of the spleen in host defense and chronicity in quartan malaria, the course of Plasmodium inui infection was studied in 39 intact rhesus monkeys, 16 monkeys splenectomized before infection, and 22 monkeys splenectomized after infection. Splenectomized animals consistently had peak parasitemias greater than 10-fold higher than those of intact animals, but the mortality rate at peak parasitemia was only higher in monkeys splenectomized before infection. Whereas intact animals had persistent infections for one to 13 years, splenectomized animals achieved self-cure (persistently negative blood smears for three or more months) within less than a year. These observations suggest that the spleen exerts a protective role during the acute stage and a suppressive role during the chronic stage of quartan malarial infection.     

The use of fluorescent nuclear dyes for the study of blood vessel structure and function: novel applications of existing techniques.

We have used nuclear fluorescent dyes to develop a technique for the study of vascular structure and function. Nuclear stained blood vessels, viewed with the appropriate filter sets, can be studied in great detail. Only the nuclei of the cells which form the walls are visible and so their positions relative to one another as well as their viability can be quickly assessed. The dyes are not toxic, therefore when the vessel contracts or relaxes, the changes in position of the nuclei can be monitored. In this paper we describe two original applications of fluorescent nuclear dyes in vascular research.     

Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.