Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: inhibitory effects and occurrence in A7r5 cells

Major sites for Rho-kinase on the myosin phosphatase target subunit (MYPT1) are Thr695 and Thr850. Phosphorylation of Thr695 inhibits phosphatase activity but the role of phosphorylation at Thr850 is not clear and is evaluated here. Phosphorylation of both Thr695 and Thr850 by Rho-kinase inhibited activity of the type 1 phosphatase catalytic subunit. Rates of phosphorylation of the two sites were similar and efficacy of inhibition following phosphorylation was equivalent for each site. Phosphorylation of each site on MYPT1 was detected in A7r5 cells, but Thr850 was preferred by Rho-kinase and Thr695 was phosphorylated by an unidentified kinase(s).     

The results of a 5-year study of listeriosis in Ukraine

Data on the isolation of Listeria spp. cultures on the territory of Ukraine, the frequency of Listeria contamination of alimentary raw materials and foodstuffs, the level of listeriosis morbidity, the clinical forms of the manifestation of this disease, as well as the occurrence of asymptomatic carriership among clinically healthy persons of epidemiologically significant professions, are given. The results of the study of the biological properties of 73 strains of Listeria spp., their sensitivity to antibiotics and Chlorophyllipt are presented.     

Regulatory properties of adenylyl and guanylyl cyclase in human spermatozoa

Completion of maturation of spermatozoa (capacitation) occurs in the female genital tract. As a result, spermatozoa acquire the high motility and the capability for acrosomal reaction, which determines their fertility. There are evidences that adenylyl cyclase and guanylyl cyclase signaling systems detected in human and mammalian spermatozoa are involved in these processes. The goal of the present study was characterization of these systems in human ejaculate spermatozoa (ES) and in human fertile spermatozoa (FS) isolated by a density gradient centrifugation. In FS homogenate the basal activity of the adenylyl cyclase (AC) was significantly higher as compared with ES (47 ± 5 vs. 28 ± 3 pmol cAMP/min per mg of protein). At the same time, the AC stimulatory effects of non-hormonal activators of soluble and membrane-bound forms of AC (NaHCO₃, Mn²⁺, forskolin, and non-hydrolyzable GTP analogue—GppNHp) in FS were lower as compared with ES. Isoproterenol, serotonin, PACAP-38, and, to the lesser extent, noradrenalin and adenosine stimulated the AC activity in ES. Among hormones inhibiting AC, only adenosine decreased the enzyme activity. At the same time, in FS the inhibitory AC effects of adenosine, noradrenalin, and serotonin were markedly expressed, and the stimulatory effects of these hormones were decreased or absent. The basal activity of guanylyl cyclase (GC) in ES and FS homogenates was 27 ± 3 and 21 ± 2 pmol cGMP for 1 min per 1 mg protein, respectively, and was significantly increased in the presence of 10 mM Mn²⁺. The stimulatory GC effects of natriuretic peptides—ANP and CNP, activators of receptor forms of GC, was significantly higher in ES than in FS, and the effect of ANP was more pronounced as compared with CNP. The data indicate the multiplicity of cAMP- and cGMP-dependent signaling cascades regulating fertility of human spermatozoa. We found that the sensitivity of AC and GC to hormones in the common pool of ES and in the fraction of highly motile FS isolated by centrifugation was essentially different, which is to be considered when using FS for accessory reproductive technology.     

Functional activity of thyroid gland in male rats with acute and mild streptozotocin diabetes

Type 1 diabetes mellitus (DM1) and dysfunction of the thyroid gland (TG) are the most common endocrine diseases, which are interrelated. However, the molecular mechanisms of thyroid dysfunction in DM1 and the role of adenylyl cyclase signaling system (ACSS) in this process remain poorly understood. Typically for studying etiology and pathogenesis of thyroid diseases in DM1 the models of acute DM1 induced by high doses of streptozotocin (STZ) are used. At the same time, a suitable model for this purpose is the model of mild DM1 initiated by moderate doses of STZ, which more closely resembles human DM1. The aim of this study was a comparative study of the functional state of the thyroid gland in rats with 30-day acute DM1 induced by injection of STZ at a dose of 65 mg/kg, and in rats with 30- and 210-day mild DM1 induced by three consecutive injections of STZ at medium doses (30–40 mg/kg). For this purpose in diabetic animals the levels of thyroid hormones and TSH and the functional activity of hormone-sensitive ACSS in membranes isolated from thyroid gland were studied. It was shown that in blood of rats with acute DM1 the levels of fT₄, fT₃, and tT₃ were decreased by 45, 23 and 19%, respectively, while the level of TSH did not change significantly. In rats with the 30-day mild DM1 the concentration of fT₄ was decreased by 32%, while the levels of tT₄, tT₃, and TSH were similar to that in control. In rats with prolonged mild DM1 after 150 and 210 days following the first treatment with STZ the levels of tT₄, fT₄, and tT₃ were significantly reduced, but the concentration of TSH in rats with 210-day mild DM1 was increased by 119%. The results obtained in the study of thyroid status and TSH levels in rats with prolonged mild DM1 are in good agreement with the data obtained in the study of thyroid diseases in patients with DM1. It was found that the AC basal activity in the membranes isolated from the thyroid gland of diabetic rats did not change, except for the rats with the prolonged mild DM1 where this activity was increased by 21%. In all groups of diabetic rats the decrease of AC stimulating effects of GppNHp (10^?5 M) and TSH (10^?8 M) was found, and in the rats with prolonged mild DM1 the AC effect of PACAP-38 (10^?6 M) was also reduced. The decrease of AC effect of TSH varied among different groups of the diabetic animals: in the rats with acute DM1 this effect was reduced by 46% and in the rats with 30- and 210-day mild DM1-by 18 and 34%. Thus, it was concluded that the key cause of the thyroid resistance to TSH under conditions of DM1 is a weakening of the signal transduction generated by TSH via the ACSS.     

Extrasynaptic membrane trafficking regulated by GluR1 serine 845 phosphorylation primes AMPA receptors for long-term potentiation

Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-D-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP.     

The dual regulation by glucagon of the adenylate cyclase system in the embryonic chick heart]

Taken in physiological concentrations, glucagon increases the activity of adenylate cyclase from the heart of 11-day chick embryos, i.e. at the earliest investigated stage. High glucagon concentrations inhibit the enzyme from cardiac membranes at all ontogenetic stages except mature chicks in which glucagon produces stimulating effect. Guanine nucleotides potentiate this effect up to the 16th day of incubation, this effect being absent at later periods. Reconstruction of adenylate cyclase system from the heart of 16-day embryos with N-proteins from mature liver tissue of chicks results in the recovery of potentiating effect. However, at later developmental stages, potentiation was absent even in the presence of N-proteins.     

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian <Emphasis Type="Italic">Dileptus anser</Emphasis>

In some unicellular eukaryotes, cAMP performs functions not only of the secondary messenger, but also of hormone, the primary messenger. We have found that cAMP is bound to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and the enzyme, adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K _D = 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N⁶,O^2′-dibutyryl-cAMP, a non-hydrolyzed cAMP analogue, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated by the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.