The similarity of the primary structure and homology of rhodopsin, beta-adrenoreceptor and muscarinic cholinoceptor

Computer analysis has been made of the primary structure of 6 different types of receptor proteins: rhodopsin, adrenoreceptor, muscarinic acetylcholine receptor, insulin receptor, nicotinic cholinoreceptor, and bacteriorhodopsin. The aim of the present investigation was to elucidate, at least partially, to what extent insignificant similarity in the primary structure of rhodopsin, muscarinic cholinoreceptor and adrenoreceptor is due to divergent, but not convergent, evolution. Nicotinic cholinoreceptor, bacteriorhodopsin and insulin receptor were chosen for comparison with rhodopsin, adrenoreceptor and muscarinic cholinoreceptor since each of these proteins exhibits this or that structural or functional property which is common for rhodopsin, adrenoreceptor or muscarinic cholinoreceptor; on the other hand, nicotinic cholinoreceptor, bacteriorhodopsin and insulin receptor differ from other receptor proteins by their molecular mechanisms. Comparison of the primary structure of rhodopsin, adrenoreceptor and muscarinic cholinoreceptor on the one hand, and insulin receptor, nicotinic cholinoreceptor and bacteriorhodopsin on the other indicates that only the former exhibit similar primary structure, whereas insulin receptor, nicotinic cholinoreceptor and bacteriorhodopsin show no similarity neither in their primary structure, nor in the primary structure of rhodopsin and other receptor proteins which are similar to the latter with respect to their mode of action. The data obtained indicate that similarity in the primary structure between rhodopsin, muscarinic cholinoreceptor and adrenoreceptor is a consequence of divergent, not convergent, evolution; in other words, these receptor proteins are homologous.     

Differential dynamics of receptor down-regulation and tyrosine aminotransferase induction following glucocorticoid treatment

Autoregulation of glucocorticoid receptor (GR) concentration in vivo may be an important determinant of steroid sensitivity. The dynamics of GR regulation were assessed and compared to regulation of tyrosine aminotransferase (TAT) expression in liver tissue taken from rats treated with a single 50 mg/kg i.v. dose of methylprednisolone. Plasma methylprednisolone concentrations were determined by HPLC analysis. Receptor and TAT message levels were determined by quantitative Northern hybridization. Methylprednisolone plasma kinetics showed a half-life of 0.6 h. Receptor occupancy occurred rapidly and cytosolic GR reappeared over 2–12 h. TAT activity rose between 2 and 6 h and then dissipated. Reduction in receptor mRNA levels occurred very rapidly, being detectable by 30 min following steroid administration. A down-regulated steady-state in GR message expression was reached by 2 h post-injection, and was maintained throughout the 18 h examined in this study. Comparison of methylprednisolone kinetics demonstrated that down-regulation was maintained long after drug was eliminated. In contrast, TAT message induction occurred with a sharp peak; maximal induction occurred between 5–6 h and return to baseline at approx. 8–10 h post-induction. This study shows that unlike TAT induction, GR message repression in vivo does not require continual presence of hormone.     

Effect of piracetam on the habituation of the neuronal cholinoreceptor membrane of the edible snail

Piracetam is shown to modulate the habituation pattern of cholinoreceptor neuronal membrane in Helix lucorum. Piracetam (10(-2) M) intensified the reversible decrease of depolarization and spike discharge induced by the local microionophoretic rhythmical application of acetylcholine to the neuron soma. The ability of piracetam to intensify the habituation of neuronal cholinoreceptor membrane may serve as a model of its facilitating effect on the development of habituation of behavioural reactions. Piracetam (5.10(-2) M) was shown to induce a shift in the membrane potential towards depolarization in the majority of identified neurons studied.     

Ageing and the Regulation of Cell Activities during Exposure to Cold

The inability to maintain body temperature and a selective pattern of changes in the regulation of cell activities were revealed by briefly exposing ageing C57B1/6J male mice to cold (10°C). The induction of liver tyrosine aminotransferase (TAT) during exposure to cold (a gene-dependent process) was markedly delayed in senescent mice (26 months old) as compared with younger mice (3–16 months old); after the delay, the rate of increase of TAT was similar to that prevailing in younger mice. Direct challenge of the liver with injections of corticosterone or insulin elicited the induction of TAT on an identical time course in young and senescent mice. These experiments provide an example of an age change in a gene-dependent cell process (the delayed induction of TAT in senescent mice during exposure to cold) which is not due to a change in the potential of the genome for responding when exogenous stimulae are supplied (injection of hormones). In contrast to the age-related change in liver cell activities, no significant changes were found in the secretion of corticosterone during exposure to cold. Although the seat of these selective age-related changes in the regulation of cell activities remains unclear, it is argued that generalized damage to the genome of cells throughout the body is not involved. The results of this and other studies showing the selective effect of age on cell activities are considered in terms of the concept that many cellular age changes represent the response of cells to primary age-related changes in humoral factors in the internal environment of the body.     

Unlinked regulation of the sensitivity of primary glucocorticoid-inducible responses in mouse mammary tumor virus infected Fu5-5 rat hepatoma cells

The enzyme tyrosine aminotransferase (TAT) is induced by unusually low concentrations of glucocorticoids in Fu5-5 cells. We have isolated clones of Fu5-5 cells infected with mouse mammary tumor virus (MMTV) in order to simultaneously compare the glucocorticoid regulation of the host cell gene, TAT, with that of another primary inducible gene, MMTV. In the two clones that were examined in detail, MMTV RNA induction occurred at 4- to 11-fold higher concentrations of dexamethasone than those needed for induction of TAT mRNA. Furthermore, the amount of agonist activity displayed by the irreversible antiglucocorticoid dexamethasone 21-mesylate was greater for the induction of TAT mRNA than for MMTV RNA. These results extend our previous observations of unequal sensitivity of induction of TAT enzyme activity in two hepatoma cell lines and show that differential glucocorticoid regulation of gene induction within the same cell can occur at a pretranslational step. The present data also indicate that the unusual properties of TAT gene induction are not shared by all primary, glucocorticoid-inducible responses of the same cell and imply that additional factors mediating differential regulation of glucocorticoid-responsive genes are involved.     

Response of rats to the presence of stressed conspecifics as a function of time of day

The physiological response of nonrestrained rats to the presence of immobilized conspecifics during the beginning of the active period and the inactive period was studied. In immobilized animals concentrations of serum corticosterone (SCS), serum glucose, and liver glycogen, and the activity of liver tyrosine aminotransferase (TAT) during both the active and the inactive periods, were consistent with earlier studies. In nonrestrained rats the presence of immobilized conspecifics induced a significant increase in SCS during the active period, whereas it had no effect during the inactive period. The level of TAT was significantly elevated in the nonrestrained rats only during the inactive period and remained unchanged during the active period. The results demonstrate a physiological influence of stressed rats on unstressed conspecifics and provide evidence for regulation of TAT activity that is dependent on the situation and the time of day.     

The reaction of the N-cholinoreceptors of the isolated neuron of the mollusk Planorbarius corneus to polymethylene-bis-trimethylammonium compounds

Experiments have been made on isolated giant neurones of the mollusc Planorbarius corneus using clamp technique at temperatures 10 and 20 degrees C. The effect of polymethylene-bis-trimethylammonium compounds with 7-18 methylene groups in the molecule (C7...C18) on N-cholinoreceptors with chloride ionic channels was investigated. All these drugs were found to be agonists. Their cholinomimetic activity depends on the number of methylene groups (up to a certain extent) in their structure. This finding stands true also for skeletal muscles of frog and chick, as it had been shown in our earlier experiments. Analysis of membrane current fluctuations showed that the elementary current, the channel opened time, temperature coefficient (Q10) of the neuronal response to application of an agonist and the calculated Q10 of the reaction rate of the agonist with cholinoreceptor did not significantly differ for C8...C18 from the reaction rate of the agonist with cholinoreceptor. As compared with C8, C12...C18 exhibited 30 ... 40 times higher cholinomimetic activity, all other parameters in them being similar. Presumably, this difference is explained by concentrating capacity of C12...C18 at the membrane site because of their higher hydrophobic properties.     

Pharmacologic properties of cholinoreceptor antibodies

The study of pharmacological properties of cholinoreceptor antibodies is of definite interest due to the possibility of simulation of some myasthenic conditions. In 1986 the authors obtained antibodies by means of immunization of rabbits with antigenic material from skeletal muscles of the extremities of Balb/c mice. It was shown that incubation of the muscle with cholinoreceptor antibodies decreased its contraction under the effect of various concentrations of acetylcholine.     

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: (1) the concentration of calcium, (2) the dish-coating material, and (3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic micotubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 μM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function. J. Cell. Physiol. 175:41–49, 1998. © 1998 Wiley-Liss, Inc.     

New trends in the field of coagulation diagnosis--new possibilities to improve monitoring of antithrombotic therapy

Two specific and sensitive enzyme immunoassays have been developed for the measurement of TAT and PTF, respectively. The TAT-ELISA uses two different antibodies binding selectively to the corresponding antigen moieties of TAT; anti-PTF antibodies were obtained from rabbits using a synthetic peptide from the COOH-terminus of PTF. Concentration in plasma samples of healthy individuals was found to be 1.45 +/- 0.4 micrograms/l for TAT, and 0.65 +/- 0.2 nMol/l for PTF. Patients with coagulation disorders showed markedly increased concentrations of both TAT and PTF. It can be assumed that these parameters might be suitable indicators for monitoring of both anticoagulant and thrombolytic therapy.     

Isolation of thermophilic ammonium-tolerant bacterium and its application to reduce ammonia emission during composting of animal wastes

A thermophilic bacterium, strain TAT105, was isolated from compost made of animal wastes. TAT105 had high tolerance to ammonium nitrogen up to 1200 mM, and highly assimilated nitrogen during the growth on swine feces. The strain was classified into Bacillus, close to Bacillus pallidus. To evaluate the effect of adding TAT105 to ammonia (NH3) emission during the composting process of animal wastes, laboratory scale composting was done. NH3 emission tended to be lower and nitrogen loss was smaller in the TAT105-added material than in the control material to which TAT105 was not added. Thermophilic ammonium-tolerant bacteria in the TAT105-added material increased to about 8x10(9) CFU/g of dry matter on the average during the tests, and most of them were judged to be TAT105 from morphological colony discrimination. These results suggested the possibility of reducing NH3 emission from composting of animal wastes by adding TAT105.     

Characterization of tyrosine aminotransferase by an immunoadsorption technic. Application to the measurement of the specific messenger RNA

A specific antiserum was used in an enzyme-linked immunoadsorbent assay (ELISA) for tyrosine amino-transferase TAT. Protein A bound on Sepharose was allowed to react with antiserum preincubated with the enzyme. Inhibition curves in the presence of protein A were parallel to those obtained in the absence of protein A. In the case of cell-free synthesized TAT, the complex bound to the solid phase contains the (35S) labelled enzyme; the sensitivity of the test was greatly increased when the bulk of protein was discarded by pretreatment of the reaction mixture at 70 degree and chromatography on DEAE cellulose. The immunoadsorbed polypeptides were analyzed by dodecylsulfate/polyacrylamide gel electrophoresis. The pattern of polypeptides neosynthesized using RNA from different origins (rat liver, hepatoma cells) and after various treatments (glucocorticoid hormones, sodium butyrate) exhibited some different in the TAT region which can be related to the level of the specific mRNA for TAT. This method is very useful for further studies on TAT gene expression and might also shed light on the mechanism of hormonal action and drug processes.     

Autogenic regulation of the sensitivity of liver cells to glucocorticoids during prolonged hormonal stimulation

The mechanisms of reversible decrease of hormone-dependent induction of tyrosine aminotransferase (TAT) by rat liver cells after prolonged administration of the glucocorticoid was studied. It was shown that the main links of the glucocorticoid action mechanism (i.e., the formation of a cytoplasmic hormone-receptor complex and the hormone accumulation in the nuclei) do not change under these conditions. It was found also that one of the necessary prerequisites for the decrease of the hormone-dependent induction of TAT is the constant production by liver cells of large amounts of TAT irrespective of whether this process is induced by the glucocorticoid or by a non-hormonal inducer, e.g., tryptophan. Using the dot-hybridization technique, it was demonstrated that the inhibition of hormone-dependent induction of TAT is correlated with the reduction of mRNA TAT. It was supposed that the main links in the mechanism of inhibition of the hormone-dependent induction are the formation of a large excess of the inducible protein--TAT--in the cells as well as the accumulation of end products of the TAT-catalyzed transamination reaction which cause a feed-back repression of the de novo synthesis of TAT. Studies with cell cultures of Morris hepatoma which is known to be sensitive to glucocorticoids revealed the ability of glucose, the end product of gluconeogenesis reactions, to provide for selective inhibition of the hormone-induced accumulation of mRNA TAT in hepatoma cells.     

Factors affecting the cholinomimetic activity of alkyltrimethylammonium compounds in experiments on isolated neurons of the mollusk Planorbarius corneus

It has been shown that the elementary current is independent whereas the duration of channel opening is slightly dependent on the number of methylene groups (from 1 to 9) in the molecule of alkyltrimethylammonium compounds. However, substances with more than 4 methylene groups exhibit lower cholinomimetic activity (i.e. the ability to increase the membrane current) and higher values of Q10 for the reaction with cholinoreceptor. It is suggested that lower activity of these compounds is due to a low rate of formation of a complex with cholinoreceptor because of the higher potential energy barrier.