Proteolysis Coupled to ATP Hydrolysis: Regulation of the Activity of Proteolytic Sites of Lon Protease from Escherichia coli

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP–Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

Trace amines and Tourette's syndrome

There has been considerable interest in recent years in possible neurochemical abnormalities in Tourette's Syndrome (TS). In studies combining neuropsychological and neurochemical measurements, we have investigated the possible roles of trace amines in this disorder. Urinary levels of free -phenylethylamine (PEA) and plasma levels of its precursor amino acid phenylalanine were decreased in TS patients when compared to values in normal children. These urinary PEA levels in TS patients were inversely related to several scores from the Tourette's Syndrome Global Scale (TSGS). Further investigation of the group of subjects with low urinary PEA indicated that they also had low levels of MHPG, normetanephrine, 5-HT andm- andp-tyramine. Patients with low PEA were also compared on an extensive battery of neuropsychological measures and observed to perform significantly worse than TS patients with normal urinary PEA levels. Biochemical measurements also suggest a possible abnormality in tryptamine turnover in TS since urinary levels of indole-3-acetic acid (IAA; the acid metabolite of tryptamine) are significantly lower in TS patients than in normal controls.     

The [3H]Tryptamine Receptor in Human Brain: Kinetics, Distribution, and Pharmacologic Profile

Abstract: The kinetics and distribution of [³H]tryptamine binding sites in human brain were investigated. Specific [³H]tryptamine binding in frontal cortex was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. A heterogeneous distribution for this binding site was demonstrated, with the highest density observed in hippocampus, thalamus ≫ caudate nucleus, frontal cortex, pons, temporal cortex > globus pallidus/putamen, cerebellum. The similarities in kinetics and distribution of the [³H]tryptamine binding site in human and rat brain indicate that these two binding sites represent homologous structures. However, the present displacement studies using various ligands (indoleamines and other tryptophan metabolites, phenylethylamines, and miscellaneous drugs) and salts (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺) indicate stereospecific displacement as well as a rank-order potency profile that is different from that reported for the rat [³H]tryptamine binding site. This suggests the presence of distinct species-dependent [³H]tryptamine binding site subtypes. Taken together with the documented electrophysiological and behavioral evidence of tryptamine-mediated effects in the rat and the recent report of a significant loss of these binding sites in human portal systemic encephalopathy, as well as the present demonstration of an effect of guanine nucleotides on [³H]-tryptamine binding affinity, these findings suggest that these binding sites might be functional receptors. The implied role of tryptamine in neuropsychiatric disorders is supported by this demonstration of a receptor for [³H]-tryptamine in human brain.     

Brain injury: New insights into neurotransmitter and receptor mechanisms

The studies reviewed here represent a continuing search for mechanisms which play a role in neurological disturbances resulting from brain injury. Focal cortical freezing lesions in rats were shown to cause a widespread decrease in local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere and this was interpreted as reflecting a depression of cortical activity. Such an interpretation was supported by the finding that in lesioned brain reduction of cerebral metabolism by pentobarbital and isoflurane was limited by the metabolic depression that has already occurred as a result of injury and by the demonstration that the energy status and substrate (glucose) supply in the cortical areas in the injured brain have not been compromised at the time when LCGU was decreased. Both the serotonergic and the noradrenergic neurotransmitter systems were implicated in functional alterations associated with injury. Cortical serotonin (5-HT) metabolism was increased throughout the lesioned hemisphere and complete inhibition of 5-HT synthesis withp-chlorophenylalanine ameliorated the decrease in cortical LCGU, interpreted as reflecting cortical functional depression. Cortical norepinephrine metabolism was bilaterally increased in focally injured brain, while prazosin, a selective ₁-noradrenergic receptor blocker, normalized cortical LCGU in the lesioned hemisphere. Low-affinity in vivo binding of [¹²⁵I]HEAT, another selective ₁-receptor ligand, was specifically increased in cortical areas of the lesioned hemisphere at the time of the greatest depression in LCGU, suggesting that ₁-adrenoreceptors may be of functional importance in injured brain. The general conclusion from this series of studies on mechanisms underlying functional disturbances in injured brain is that both the serotonergic and the noradrenergic neurotransmitter systems are involved in the widespread cortical depression which develops with time as a consequence of a focal lesion. The data are compatible with the inhibitory effects of NE and 5-HT in the cortex and with the hypothesis that these two transmitter systems affect cortical information processing.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

Neuropeptides in interneurons of the insect brain

A large number of neuropeptides has been identified in the brain of insects. At least 35 neuropeptide precursor genes have been characterized in Drosophila melanogaster, some of which encode multiple peptides. Additional neuropeptides have been found in other insect species. With a few notable exceptions, most of the neuropeptides have been demonstrated in brain interneurons of various types. The products of each neuropeptide precursor seem to be co-expressed, and each precursor displays a unique neuronal distribution pattern. Commonly, each type of neuropeptide is localized to a relatively small number of neurons. We describe the distribution of neuropeptides in brain interneurons of a few well-studied insect species. Emphasis has been placed upon interneurons innervating specific brain areas, such as the optic lobes, accessory medulla, antennal lobes, central body, and mushroom bodies. The functional roles of some neuropeptides and their receptors have been investigated in D. melanogaster by molecular genetics techniques. In addition, behavioral and electrophysiological assays have addressed neuropeptide functions in the cockroach Leucophaea maderae. Thus, the involvement of brain neuropeptides in circadian clock function, olfactory processing, various aspects of feeding behavior, and learning and memory are highlighted in this review. Studies so far indicate that neuropeptides can play a multitude of functional roles in the brain and that even single neuropeptides are likely to be multifunctional.The original research in the authors’ laboratories was supported by DFG grants HO 950/14 and 950/16 (U.H.) and Swedish Research Council grant VR 621-2004-3715 (D.R.N).     

Autoradiographic demonstration of α2 adrenoceptors in the bovine neurohypophysis

Summary Autoradiography of sections from neurohypophyses treated with tritiated clonidine has shown specific binding to ₂-adrenoceptor sites in the neurohypophysis but not in the intermediate lobe. Other studies have shown that ₂ agonists such as clonidine can cause a fall in circulating antidiuretic hormone; it is therefore possible to speculate that this action could be a direct one on the neurohypophysis since the appropriate binding sites have been shown to exist.     

Comparison of melatonin-binding sites in the brain of two amphibians: an autoradiographic study

Neuroanatomic comparison of the binding capability of 2-[¹²⁵I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[¹²⁵I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.     

Mas-allatotropin/Lom-AG-myotropin I immunostaining in the brain of the locust, <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

Mas-allatotropin (Mas-AT) and Lom-accessory gland-myotropin I (Lom-AG-MTI) are two members of a conserved family of insect neuropeptides, collectively termed allatotropins, which have diverse functions, ranging from stimulation of juvenile hormone secretion to myotropic effects on heart and hindgut. In addition, allatotropins appear to be abundant within the nervous system, suggesting neuroactive roles. To identify neurons in the insect brain suitable for a neurophysiological analysis of the roles of allatotropins, we used antisera against Mas-AT and Lom-AG-MTI to map allatotropin-immunoreactive neurons in the brain of a suitable insect, the locust Schistocerca gregaria. Both antisera revealed basically identical staining patterns throughout the locust brain with more than 12,500 immunostained interneurons per brain hemisphere. Neurosecretory cells were not labeled, and the retrocerebral complex was devoid of immunostaining. Prominent immunoreactive cell types include about 9,600 lamina monopolar neurons, medulla to lobula interneurons, local neurons of the antennal lobe, a giant interneuron of the mushroom body, projection neurons of the glomerular lobe to the mushroom body, and three systems of tangential neurons of the central complex. Several groups of neurons showed colocalization of Mas-AT- and -aminobutyric acid immunostaining. Mass spectrometric analysis identified a peptide with a molecular mass identical to Lom-AG-MTI in all major parts of the locust brain but not in the retrocerebral complex. This study strongly suggests that Lom-AG-MTI is highly abundant in the locust brain, and is likely to play a neuroactive role in many brain circuits including all stages of sensory processing, learning and memory, and higher levels of motor control.This work was supported by DFG grant HO 950/14 to U.H.     

Rate of polymer formation and entropy production during competitive replication

Summary The rate of increase in the mean polymer formation rate constant during competitive replication byQ RNA variants (Kramer et al., 1974) has been shown to agree statistically with the variance in their formation rate constants. This result demonstrates that Fisher's fundamental theorem of natural selection (Fisher, 1930) can define time variations in the mean rate of synthesis for a heterogeneous population of replicating polymers. It was also revealed that RNA replication, far from equilibrium, accompanied a progressive decrease in the order of the entropy production derivative, with respect to time, that reached a maximum (with the next higher order being zero). Maximization of entropy at equilibrium, in compliance with the second law of thermodynamics, therefore appears as a natural extension of the earlier non-equilibrium pattern of entropy production within the system. The order of the zero-valued entropy production derivative was shown to be determined by the chemical affinity, and its rate of decrease was specified by the mean polymer formation rate constant.     

Effect of monofluoromethyldopa (MFMD) on trace amine levels

The concentrations of the trace amines, m-tyramine, p-tyramine, phenylethylamine and tryptamine, were measured in the striatum of the brain and in the kidney of adult rats treated with alpha-monofluoromethyldopa (MFMD), an inhibitor of aromatic amino acid decarboxylase. While MFMD decreased the levels of all four amines in the kidney, only phenylethylamine and tryptamine levels were decreased in the striatum compared to control. Striatal p-tyramine levels were not affected, while striatal m-tyramine levels were increased by MFMD. When the rats were injected with a monoamine oxidase (MAO) inhibitor before MFMD administration, similar changes in striatal and kidney trace amine levels were observed compared to MFMD alone.     

Ethylene binding sites in higher plants

A review of work carried out on ethylene binding in higher plants is presented. The use of radio-labelled displacement assays has identified specific ¹⁴C-ethylene binding in all tissues so far studied. virtually all higher plants studied contain at least two classes of ethylene binding site, one of which fully associates and dissociates in about 2 h and a class of sites that takes up to 20 h to become fully saturated. Although the types of site differ in their rate constants of association they have similar and high affinities for ethylene.A series of Arabidopsis thaliana mutants shown to vary in sensitivity to ethylene have been analysed for ¹⁴C-ethylene binding. One mutant, eti 5, which was shown to be unaffected by ethylene concentrations of up to 10,000 L L^–1 was also shown to exhibit reduced binding. In vivo and in vitro studies on pea have shown that ethylene binding can be detected in this tissue. In vitro studies have shown that both membrane and cytosolic fractions contain measurable amounts of ethylene binding. Interestingly, cytosolic ethylene binding consisted only of the fast associating/dissociating type.Developing cotyledons of Phaseolus vulgaris contain a higher concentration of ethylene binding sites that other tissues and only contain the slow dissociating component. These facets have allowed the purification of ethylene binding protein(s) (EBP) from this tissue. The proteins which bind ethylene can be resolved into two bands of 26 and 28 kDa on semi-denaturing PAGE and the proteins appear to be single entities on a 2-D gels.Data will be presented which indicate a possible role for heterotrimetric G-proteins in the early stages of the ethylene signal transduction pathway.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Effect of morphine and abstinence syndrome on [3H]bromoxidine binding to α2-adrenoreceptors in rat brain

At 4 days after the implantation of two subcutaneous 75 mg morphine pellets in the back skin, rats were morphine-dependent. In the three layers studied in the occipital cortex we found that the values of the ₂-adrenergic agonist [³H]bromoxidine binding increased with respect to animals implanted with placebo pellets. Typical behavioral and physiological symptoms of the abstinence syndrome appeared 30 minutes after administration of naloxone, [³H]bromoxidine binding values being similar to those obtained in animals implanted with placebo pellets. The pattern of response of the [³H]bromoxidine binding was similar in the hippocampus and the superficial gray layer of the superior colliculus of the mesencephalon, but the differences were not statistically significant in these areas. This paper concludes that exist brain regional differences in the ₂-adrenoreceptors response under morphine-treatment and possibly under naloxone-induced morphine abstinence syndrome.     

An autoradiographic study of α1-adrenergic receptors in the brain of the Japanese quail (Coturnix coturnix japonica)

Summary The neuroanatomical distribution of ₁-adrenergic receptors was studied in Japanese quail by quantitative in vitro autoradiography using the specific antagonist [³H]prazosin as the ligand. The presence of saturable (B_max <200 fmol/mg protein) high affinity (K_d < 0.12 nM) binding sites was detected by saturation analysis. High concentrations of [³H]prazosin binding sites were detected in the archistriatum/pars ventralis, the hippocampus, the cortex piriformis, the area corticoidea dorsolateralis, the dorsal thalamus, and the nucleus praetectalis. Lower concentrations were seen in the intercollicular nucleus, the lateral septum, and the posterior and tuberal hypothalamus. Very little binding was seen in the preoptic and anterior hypothalamic areas. The relatively high number of binding sites identified in the telencephalic structures agrees well with previous mammalian studies. This is in contrast with the pattern in the anterior hypothalamus where, in mammals, a number of nuclei have been reported to contain a high receptor density.     

Characterization of Human α4β2 Neuronal Nicotinic Receptors Stably Expressed in SH-EP1 Cells

These studies characterized human alpha4beta2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in transfected SH-EPI-halpha4beta2 cells were functional, as determined by increases in intracellular Ca2+ in response to a nicotine stimulus. Nicotine increased Fura-2 fluorescence in a concentration-dependent manner with an apparent EC50 of 2.4 microM, a response that was blocked by the specific antagonist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca2+ response inactivated by 44%, an effect that recovered within 24 hours. SH-EP1-halpha4beta2 cells expressed a single class of high affinity binding sites for [3H]cytisine with a Kd of 0.63 +/- 0.08 nM and a Bmax of 6,797 +/- 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotine for 24 hours increased the Bmax by 45% without changing affinity, a concentration-dependent effect with an EC50, of 58.6 nM. The nicotine-induced up regulation was reversible, and control values were achieved within 24 hours. Results indicate that SH-EPI-halpha4beta2 cells may be a good model system to study regulation of human alpha4beta2 receptors, the most abundant nicotinic receptor subtype in brain.     

Effects ofp-chloroamphetamine on brain serotonin neurons

p-Chloroamphetamine (PCA) is a useful pharmacologic tool for selectively increasing brain serotonin function acutely by release of serotonin into the synaptic cleft. PCA produces behavioral, neurochemical and neuroendocrine effects believed due to serotonin release after doses in the range of 0.5–5 mg/kg. At higher doses and at longer times, PCA causes depletion of brain serotonin. The mechanisms of this depletion are not well understood but require the serotonin uptake carrier. Antagonism of PCA-induced depletion of brain serotonin is a useful means of assessing the ability of a compound to block the serotonin uptake carrier on brain serotonin neurons. PCA can also be used as a neurotoxic agent to deplete brain serotonin in functional studies, apparently by destroying some serotonergic nerve terminals. Used in this way, PCA has an advantage over 5,6- and 5,7-dihydroxytryptamines in being effective by systemic injection, and it affects brain serotonergic projections with a different neuroanatomic specificity than the dihydroxytryptamines.Special issue dedicated to Dr. Morris H. Aprison.     

[3H]norharman ([3H]β-carboline) binds reversibly and with high affinity to a specific binding site in rat liver

In addition to the known binding of norharman (NH) to monoamine oxidase (MAO) and benzodiazepine (BZ) binding sites (at M concentrations), a distinct class of high-affinity NH binding sites was discovered in rat brain (1,2). Investigations of several organs of the rat led to the discovery of high affinity binding sites in the liver, which successfully could be solubilized from P₂ membrane homogenate (0.25% w/v Triton X-100). Scatchard analysis revealed an apparent K_D value of 26±8 nM and a maximum number of binding sites of 11±3 pmol/mg protein (n=14). Association kinetics showed that equilibrium was nearly reached after two hours. Dissociaton was totally complete only after more than 16 hours. The MAO-inhibitors examined did not influence the binding characteristics. No displacement of specific binding could be found by haloperidol.