Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance.

The rapid emergence of drug resistant Staphylococcus aureus (S. aureus) poses a serious threat to public health globally. Silver (Ag)-based antimicrobials are promising to combat antibiotic resistant S. aureus, yet their molecular targets are largely elusive. Herein, we separate and identify 38 authentic Ag+-binding proteins in S. aureus at the whole-cell scale. We then capture the molecular snapshot on the dynamic action of Ag+ against S. aureus and further validate that Ag+ could inhibit a key target 6-phosphogluconate dehydrogenase through binding to catalytic His185 by X-ray crystallography. Significantly, the multi-target mode of action of Ag+ (and nanosilver) endows its sustainable antimicrobial efficacy, leading to enhanced efficacy of conventional antibiotics and resensitization of MRSA to antibiotics. Our study resolves the long-standing question of the molecular targets of silver in S. aureus and offers insights into the sustainable bacterial susceptibility of silver, providing a potential approach for combating antimicrobial resistance.

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi.

Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC50 40.5 µM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.

Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study.

The inhibitory effects of some drugs on 6-phosphogluconate dehydrogenase from human erythrocytes have been investigated. For this purpose, initially, erythrocyte 6-phosphogluconate dehydrogenase was purified 3364 times in a yield of 58% by using ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity gel. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Many commonly used drugs were investigated in this study. Some drugs (ketotifen (K(i): 8.3 +/- 1.7 microM), dacarbazine (K(i): 10.1 +/- 0.7 microM), meloxicam (K(i): 50.9 +/- 13.2 microM), furosemide (K(i): 127 +/- 37.8 microM), methotrexate (K(i): 136.7 +/- 25.3 microM), metochloropramide hydrochloride (K(i): 2.1113 +/- 0.6979 mM), ritodrine hydrochloride (K(i): 6.0353 +/- 1.2783 mM), and gadopentetic acid (K(i): 73.4 +/- 21.9 mM)) inhibited enzyme activity in vitro. K(i) constants for the enzyme were found by means of Lineweaver-Burk graphs. All drugs showed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting activity percent vs [I].

The effects of n-3 polyunsaturated fatty acids by gavage on some metabolic enzymes of rat liver.

In this experimental study, the effect of fish n-3 fatty acids was studied on the some important enzymes of carbohydrate metabolism, hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) in rat liver. Wistar albino rats of experimental group (n= 9) were supplemented fish omega-3 fatty acids (n-3 PUFA) as 0.4 g/kg bw. by gavage for 30 days in addition to their normal diet. Isotonic solution was given to the control group (n= 8) by the same way. At 30th day, the rats were killed by decapitation under ether anesthesia, autopsied and liver was removed. Spectrophotometric methods were used to determine the activities of above-mentioned enzymes in the liver. The n-3 PUFA caused increases in the activities of HK, G6PD, LDH, and MDH in comparison with control. These increases were statistically significant (P < 0.01) except 6PGD activity. As a result, n-3 PUFA may regulate the metabolic function of liver effectively by increasing HK, G6PD, 6PGD, LDH, and MDH enzyme activities of rat liver when added in enough amounts to the regular diet.

Tissue specific, sex and age--related differences in the 6-phosphogluconate dehydrogenase gene expression.

Data presented in thid paper indicate that: (1) the age-related changes in 6-phosphogluconate dehydrogenase (6PGDH) activity depend on sex and tissue. No differences in the liver 6PGDH activity between young (1-month-old) males and females were found. In adult males, the activity was the same as in young animals but, in adult females, it reached the value twice as high as in the young. In adipose tissue (both white and brown) and kidney cortex, the enzyme activity decreased with age both in males and females. There were no differences between males and females 6PGDH activity in brain, heart and skeletal muscle. (2) The sex and age-related changes in the liver 6PGDH activity occur predominantly at the level of mRNA cellular concentration. (3) In the liver of ovariectomized rats decrease of 6PGDH activity and mRNA level was observed. Oestradiol administration to ovariectomized rats restored liver 6PGDH activity and liver 6PGDH mRNA levels to that observed in controls. No changes in 6PGDH activity and mRNA levels were found in white adipose tissue (WAT) of ovariectomized adult rats and in ovariectomized rats treated with oestradiol. (4) Oestradiol administration to males caused an increase of liver 6PGDH activity and mRNA levels to values observed in females, but was without an effect on WAT 6PGDH activity and mRNA level. (5) These results suggest that 6PGDH activity in different tissues is not regulated in coordinate fashion and that oestradiol plays an important role in the liver enzyme activity regulation.

Neurotoxin 6-aminonicotinamide affects levels of soluble proteins and enzyme activities in various tissues of golden hamsters.

The effects of neurotoxin 6-aminonicotinamide (6-AN) on the levels of soluble proteins and enzyme activities in various tissues of golden hamsters were investigated. SDS-polyacrylamide gel electrophoresis showed that a soluble spinal cord protein with molecular mass 75.0 kDa was present at a higher concentration in the treated group compared to that in the control while that of a molecular mass 64.8 kDa appeared to be missing. However, there were no noticeable differences in protein concentrations observed with the cerebrum, brain stem, and cerebellum. Similarly, treatment with 6-AN decreased the concentration of a soluble protein in pectoral muscle having molecular mass 97.2 kDa and increased those having molecular masses 207.4 and 32.1 kDa. In the kidney, soluble proteins with molecular masses 176.6 kDa was missing and those of molecular masses 97.6, 49, 43.3, and 33.8 kDa were decreased whereas those of molecular masses 64.7 and 33.1 kDa were increased. In the testis the soluble proteins with molecular masses 125.4, 88.7, 69.0, 31.2, 19.1, and 17.4 kDa were missing and those of molecular masses 97.0, 51.3, 42.0, 33.0, 27.2, and 22.6 kDa were present in lower amounts whereas those of molecular masses 311.5, 75.0, 64.0, 54.1, and 53.2 kDa were present in higher amounts. The specific activity of 6-phosphogluconate dehydrogenase was markedly increased in the liver but that of other tissues was not affected. Acetylcholinesterase activity was markedly reduced in the spleen but was enhanced in the intestine. Monoamine oxidase activity was markedly reduced in the brain stem, cerebrum, kidney, and liver. The results suggest that the changes in levels of soluble proteins and enzyme activities shown with golden hamster tissues by 6-AN administration were quite different from those shown with quail tissues.

Effects of NAD or NADP on the stability of liver and pectoral muscle enzymes in 3-acetylpyridine treated quail by heat and trypsin.

(1) The effects of long term treatment with 3-acetylpyridine on the stability of enzymes towards heat and trypsin treatment were studied. (2) In the liver NAD or NADP provided a similar degree of protection against heat inactivation at 55 degrees C for 6-phosphogluconate dehydrogenase (24%), glyceraldehyde-3-phosphate dehydrogenase (24%) and malic enzyme (20%), low level of protection of lactate dehydrogenase (13%) but didn't affect acetylcholinesterase at all. In the muscle, however, there was substantial protection against heat inactivation by coenzyme of glyceraldehyde-3-phosphate dehydrogenase (52%), an intermediate level of protection of lactate dehydrogenase (25%), low level of protection of 6-phosphogluconate dehydrogenase (17%) and malic enzyme (17%) and almost no protection of acetylcholinesterase. (3) In the susceptibility towards trypsin a low but similar degree of protection for dehydrogenases by coenzymes was observed in the liver whereas in the muscle there was substantial protection against trypsin inactivation by NAD of glyceraldehyde-3-phosphate dehydrogenase, an intermediate level of protection of 6-phosphogluconate dehydrogenase and malic enzyme and very little protection of lactate dehydrogenase but no protection of acetylcholinesterase. Among enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against heat and trypsin inactivation by NAD. (4) The results suggest that the effect of 3-acetylpyridine treatment on the stability of muscle glyceraldehyde-3-phosphate dehydrogenase appears to be quite specific and selective.

Inactivation and cleavage of liver 6-P-gluconate dehydrogenase during irradiation in the presence of vanadate.

Lamb liver phosphogluconate dehydrogenase is inactivated and selectively cleaved during irradiation in the presence of vanadate. Under our experimental conditions, the correlation between the species of vanadate in solution and rates of enzyme inactivation and cleavage indicates tetravanadate as the most likely photosensitizing agent, in agreement with previous data on other proteins. The enzyme is inactivated more rapidly at acidic pH and is partially protected by the coenzyme NADP, but not by the substrate phosphogluconate. Complete inactivation is obtained when only half of the protein is cleaved into smaller peptides. Differences in the pattern of the peptides produced are observed when irradiation is carried out in phosphate rather than in Hepes buffer: in the former instance cleavage results into formation of a main peptide of 47 kDa, while in latter case two additional peptides of 31 and 25 kDa are produced.

[The effect of thyroid hormones and 5-azacytidine on the gene transcription of malic enzyme and 6-phosphogluconate dehydrogenase]. / Vliianie tireoidnykh gormonov i 5-azatsitidina na transkriptsiiu genov malik-fermenta i 6-fosfogliukonatdegidrogenazy.

The effect of thyroid hormones and the methylation inhibitor 5-azacytidine on the level of expression of thyroid hormone-responsive genes (malic enzyme and 6-phosphogluconate dehydrogenase genes) has been studied. DNA-RNA hybridization has shown there is an inverse correlation between the level of DNA methylation and gene expression of malic enzyme and 6-phosphogluconate dehydrogenase. Thus it suggests that the regulation of thyroid hormone gene expression can take place via DNA methylation blocking and that DNA demethylation is part of structural changes essential to the binding of thyroid hormones with DNA elements recognized by thyroid hormone receptors and to further induction of synthesis of specific mRNA.

Subunits asymmetry in the ternary complex of lamb liver 6-phosphogluconate dehydrogenase detected by a NADP analogue.

Incubation of lamb liver 6-phosphogluconate dehydrogenase, a dimeric enzyme with periodate-oxidized NADP causes the inactivation of the enzyme due to the covalent binding of 2 mol of inhibitor/mol of dimer. In the presence of substrate, the inactivation is faster and is almost complete after the labelling of only one subunit. These results not only confirm the hypothesis of a 'half-of-the-sites' mechanism of action of the enzyme, but also suggest that the formation of the ternary complex (enzyme-substrate-coenzyme) in one subunit causes a conformational change that makes the other subunit unable to bind the coenzyme (and even the adenylic part of it) and, thus, this subunit becomes inactive. It appears that while one subunit catalyses the oxidation of 6-phosphogluconate the other is inactive in this reaction.

[Effect of sodium nitrite poisoning on the activity of enzymes of anti-oxidant protection and peroxidation processes in mouse erythrocytes]. / Vliianie intoksikatsii nitritom natriia na aktivnost' fermentov antioksidantnoi zashchity i protsessy peroksidatsii v éritrotsitakh myshi.

The intoxication of white mice with sodium nitrite results in the decrease of red cell superoxide dismutase (SOD) and catalase activity. The glutathione peroxidase activity is the same as in the control group. The level of red cell lipid peroxidation in the group of mice that receive sodium nitrite is higher as compared to the control group. After the intoxication the total activity of glucose-6-phosphate dehydrogenase and dehydrogenase of 6-phosphogluconate as well as the activity of glutathione reductase are higher than in the control group. The level of SH-groups and reduced glutathione is higher in the group of mice that receive sodium nitrite in comparison with the control group.

Effects of prolactin and androgens on enzymes of carbohydrate metabolism in prostate of castrated bonnet monkeys Macaca radiata (Geoffroy).

Effects of prolactin (PRL), bromocriptine (Br), testosterone propionate (TP), dihydrotestosterone (DHT) and the combinations of these androgens with PRL/Br on the specific activities of caudal and cranial prostatic cellular enzymes involved in carbohydrate metabolism in castrated mature bonnet monkeys have been studied. Castration decreased all the enzymes studied such as hexokinase (HK), 6-phosphofructokinase (6-PFK), glyceraldehyde-3-phosphate dehydrogenase (G-3-PD), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) in the cranial and caudal prostates. PRL elevated the activities of all the enzymes above normal except G-3-PD of cranial lobe. In the caudal lobe, PRL brought back the activities of HK, PFK, PK, G-6-PD to normal and 6-PGD above normal except G-3-PD. TP/DHT treatment increased all the enzymes in both the lobes. PRL given along with TP/DHT further enhanced the androgen action with regard to HK, PK, G-6-PD and 6-PGD of cranial and PFK, G-3-PD, PK, G-6-PD and 6-PGD of caudal lobe. Br treatment did not produce any alteration of these enzymes in both the lobes. In the cranial lobe, during Br+TP/DHT treatment, the stimulating effects of androgen were unaffected on all the enzymes except PK. On the other hand in the caudal, the stimulatory effects of androgens were affected and the activities of HK, PFK, PK and 6-PGD were significantly decreased. The present results suggest that PRL has a direct as well as a synergistic action with androgens on enzymes of EMP and HMP shunt in the prostates of monkeys.

The role of NADPH in the regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in rat adipose tissue.

Previous studies examining the regulation of the synthesis of G6PDH and 6PGDH in rat liver and adipose tissue have focused on the induction of these enzymes by different diets and some hormones. In rat liver these enzymatic activities seem to be regulated by a mechanism involving changes in the NADPH requirements. In this paper we have studied the effect of changes in the flux through different NADPH-consuming pathways on G6PDH and 6PGDH levels in adipose tissue and on the NADPH/NADP ratio. The results show that: I) an increase in the consumption of NADPH, caused by the activation of either fatty acid synthesis or detoxification systems which consume NADPH, is paralleled by an increase in the levels of these enzymes; II) when the increase in consumption of NADPH is prevented, the G6PDH and 6PGDH levels do not change.

The effect of menadione epoxide on the experimental immune arthritis in the rabbit.

It was shown previously that the experimentally induced arthritis in the rabbit can be largely nullified by subsequent treatment with menadione (by gavage). It is now shown that menadione epoxide, as is produced in the vitamin K cycle, also exerts a beneficial effect histologically and biochemically. Such treatment decreased both the glucose 6-phosphate dehydrogenase and the 6-phosphogluconolactonase activities in the synovial lining cells of the challenged joints towards values found in the unchallenged joints; it had only equivocal effects on the 6-phosphogluconate dehydrogenase activity. The results indicated that the epoxide might be interfering primarily with the lactonase activity.