Béla Tóth

Efficient inhibition of muscle and liver glycogen phosphorylases by a new glucopyranosylidene-spiro-thiohydantoin

Reaction of C-(1-bromo-1-deoxy-beta-glucopyranosyl)formamide 2 with thiocyanate ions was the key step of a short synthesis of D-glucopyanosylidene-spiro-thiohydantoin 7 which proved to be a potent inhibitor of muscle and liver glycogen phosphorylases.

Kinetic and crystallographic studies of glucopyranosylidene spirothiohydantoin binding to glycogen phosphorylase B.

Glucopyranosylidene spirothiohydantoin (TH) has been identified as a potential inhibitor of both muscle and liver glycogen phosphorylase b (GPb) and a (GPa) and shown to diminish liver GPa activity in vitro. Kinetic experiments reported here show that TH inhibits muscle GPb competitively with respect to both substrates phosphate (K(i)=2.3 microM) and glycogen (K(i)=2.8 microM). The structure of the GPb-TH complex has been determined at a resolution of 2.26 A and refined to a crystallographic R value of 0.193 (R(free)=0.211). The structure of GPb-TH complex reveals that the inhibitor can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provides a basis of understanding potency and specificity of the inhibitor. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the glucose complex, while the rigid thiohydantoin group is in a favourable electrostatic environment and makes additional polar contacts to the protein.

Gram-scale synthesis of a glucopyranosylidene-spiro-thiohydantoin and its effect on hepatic glycogen metabolism studied in vitro and in vivo

Abstract A high yielding, simple synthesis is described starting from d -glucose to produce gram quantities of a glucopyranosylidene-spiro-thiohydantoin. This compound efficiently inhibited the activity of rat liver glycogen phosphorylase a ; moreover, it also activated phosphorylase phosphatase which, in turn, decreased the amount of glycogen phosphorylase a . Both effects result in the inhibition of glycogen mobilization and the formation of glucose from glycogen.

Hormonal regulation of phosphorylase phosphatase activity in rat liver

The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat‐stable phosphatase inhibitors in the hormone‐treated samples. Liver protein kinases (regulatory subunit of cAMP‐dependent protein kinase and/or Ca2+ ‐dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A).

Effect of fructose 1-phosphate on the activation of liver glycogen synthase

The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.

Regulatory subunit of type II cAMP‐dependent protein kinase as substrate and inhibitor of protein phosphatase‐1 and ‐2A

The dissociated regulatory subunit (RII) of autophosphorylated cAMP‐dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase‐1 and ‐2A (phosphatase‐1c and ‐2Ac) and by a high‐M r, polycation‐dependent form of phosphatase‐2A (2A0) with K m values of 5,0.3 and 1 μM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase‐1c, whereas polycations (histone H1 or polybrene) markedly stimulated phosphatase‐2Ac and ‐2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half‐maximum inhibitory concentrations of 0.1–0.36 μM.

Heterotropic interactions of AMP and glucose binding sites in phosphorylase a are destroyed by limited proteolysis.

Subtilisin BPN hydrolyses a single peptide bond in phosphorylase a. The two proteolytic fragments are attached to each other by noncovalent bonds in solution as shown by gel filtration and ultracentrifugation studies. The subtilisin nicked phosphorylase a is inactive, however, still binds AMP and glucose as judged by equilibrium dialysis and fluorescence experiments. The modified enzyme can be dephosphorylated by protein phosphatase and AMP is an effective inhibitor of the dephosphorylation reaction. Glucose cannot cancel the AMP inhibition as well as cannot expel AMP from the nucleotide binding site. Thus a single nick in the polypeptide chain breaks the communication between the two ligand binding domains.

Interaction of ligands in phosphorylase A as monitored by crosslinking and enzymatic modifications: Synergism of glucose and caffeine manifested in the exposure of N-terminal segment

1. Glycogen, caffeine and glucose dissociate phosphorylase a tetramer to dimers with half-maximum effect at 0.16%, 1.1 and 71 mM concentration, respectively, as monitored by crosslinking with dimethyl suberimidate at 18 degrees C. 2. The above ligands increase the rate of dephosphorylation and tryptic digestion of phosphorylase alpha at 18 degrees C in the same way with half-maximum effect at 0.04%, 0.1 and 9 mM concentration, respectively. 3. Caffeine and glucose acted synergistically in tetramer dissociation as well as in the enzymic modifications. 4. The alpha-anomer of D-glucose was twice as effective as its mutarotational equilibrium solution.

Aliphatic alcohol contaminants of illegally produced spirits inhibit phagocytosis by human granulocytes

Abstract Context: Unregulated production of spirits in many countries leads to products containing appreciable levels of aliphatic alcohols (AAs) and is the main source of human exposure to these substances worldwide. Previous studies have confirmed that alcohol abuse can lead to ethanol-induced immunosuppression and thereby increased susceptibility to infectious diseases. Granulocytes, as professional phagocytic cells, play a crucial role in engulfment and killing of pathogenic microorganisms. Thus, a decrease in their phagocytic activity has been invoked as a factor in the impaired antimicrobial defense observed in alcoholics. However, AAs consumed as contaminants of illicit spirits may also influence phagocytosis, thereby contributing to a further decrease in microbicidal activity but, so far, this has not been studied. Objective: Therefore, the aim of this study was to measure granulocyte phagocytosis following treatment of granulocytes with those higher alcohols found in illegal spirits. Materials and methods: Granulocytes were isolated from human peripheral blood. Then phagocytosis of opsonized zymosan particles by granulocytes treated with AAs individually and in combination was determined. Results: These alcohols inhibited phagocytosis in a concentration-dependent manner and at lower concentrations when combined than when tested individually. Discussion and conclusion: Due to their synergistic effects, it is possible that, in combination with ethanol, they may inhibit phagocytosis in a clinically meaningful way in episodic heavy drinkers.

Randomized clinical trial to comparing efficacy of daily, weekly and monthly administration of vitamin D3

The comparative efficacy and safety profiles of selected daily 1000u2009IU, weekly 7000u2009IU and monthly 30,000u2009IU vitamin D3—not previously investigated—will be evaluated. Here, a prospective, randomized clinical trial, comparing efficacy and safety of a daily single dose of 1000u2009IU (group A) to a once-weekly 7000u2009IU dose (group B), or monthly 30,000u2009IU dose (group C) of vitamin D3. The present study is a controlled, randomized, open-label, multicenter clinical trial, 3u2009 months in duration. Sixty-four adult subjects with vitamin D deficiency (25OHD<20u2009ng/ml), were included according to the inclusion and exclusion criteria. Dose-responses for increases in serum vitamin 25OHD were statistically equivalent for each of the three groups: A, B and C. Outcomes were 13.0u2009±u20091.5; 12.6u2009±u20091.1 and 12.9u2009±u20090.9u2009ng/ml increases in serum 25OHD per 1000u2009IU, daily, weekly and monthly, respectively. The treatment of subjects with selected doses restored 25OHD values to levels above 20u2009ng/ml in all groups. Treatment with distinct administration frequency of vitamin D3 did not exhibit any differences in safety parameters. The daily, weekly and monthly administrations of daily equivalent of 1000u2009IU of vitamin D3 provide equal efficacy and safety profiles.