Avian influenza viruses in New Zealand wild birds, with an emphasis on subtypes H5 and H7: Their distinctive epidemiology and genomic properties.

The rapid spread of highly pathogenic avian influenza (HPAI) A (H5N1) viruses in Southeast Asia in 2004 prompted the New Zealand Ministry for Primary Industries to expand its avian influenza surveillance in wild birds. A total of 18,693 birds were sampled between 2004 and 2020, including migratory shorebirds (in 2004-2009), other coastal species (in 2009-2010), and resident waterfowl (in 2004-2020). No avian influenza viruses (AIVs) were isolated from cloacal or oropharyngeal samples from migratory shorebirds or resident coastal species. Two samples from red knots (Calidris canutus) tested positive by influenza A RT-qPCR, but virus could not be isolated and no further characterization could be undertaken. In contrast, 6179 samples from 15,740 mallards (Anas platyrhynchos) tested positive by influenza A RT-qPCR. Of these, 344 were positive for H5 and 51 for H7. All H5 and H7 viruses detected were of low pathogenicity confirmed by a lack of multiple basic amino acids at the hemagglutinin (HA) cleavage site. Twenty H5 viruses (six different neuraminidase [NA] subtypes) and 10 H7 viruses (two different NA subtypes) were propagated and characterized genetically. From H5- or H7-negative samples that tested positive by influenza A RT-qPCR, 326 AIVs were isolated, representing 41 HA/NA combinations. The most frequently isolated subtypes were H4N6, H3N8, H3N2, and H10N3. Multivariable logistic regression analysis of the relations between the location and year of sampling, and presence of AIV in individual waterfowl showed that the AIV risk at a given location varied from year to year. The H5 and H7 isolates both formed monophyletic HA groups. The H5 viruses were most closely related to North American lineages, whereas the H7 viruses formed a sister cluster relationship with wild bird viruses of the Eurasian and Australian lineages. Bayesian analysis indicates that the H5 and H7 viruses have circulated in resident mallards in New Zealand for some time. Correspondingly, we found limited evidence of influenza viruses in the major migratory bird populations visiting New Zealand. Findings suggest a low probability of introduction of HPAI viruses via long-distance bird migration and a unique epidemiology of AIV in New Zealand.

No evidence of enemy release in pathogen and microbial communities of common wasps (Vespula vulgaris) in their native and introduced range.

When invasive species move to new environments they typically experience population bottlenecks that limit the probability that pathogens and parasites are also moved. The invasive species may thus be released from biotic interactions that can be a major source of density-dependent mortality, referred to as enemy release. We examined for evidence of enemy release in populations of the common wasp (Vespula vulgaris), which attains high densities and represents a major threat to biodiversity in its invaded range. Mass spectrometry proteomic methods were used to compare the microbial communities in wasp populations in the native (Belgium and England) and invaded range (Argentina and New Zealand). We found no evidence of enemy release, as the number of microbial taxa was similar in both the introduced and native range. However, some evidence of distinctiveness in the microbial communities was observed between countries. The pathogens observed were similar to a variety of taxa observed in honey bees. These taxa included Nosema, Paenibacillus, and Yersina spp. Genomic methods confirmed a diversity of Nosema spp., Actinobacteria, and the Deformed wing and Kashmir bee viruses. We also analysed published records of bacteria, viruses, nematodes and fungi from both V. vulgaris and the related invader V. germanica. Thirty-three different microorganism taxa have been associated with wasps including Kashmir bee virus and entomophagous fungi such as Aspergillus flavus. There was no evidence that the presence or absence of these microorganisms was dependent on region of wasp samples (i.e. their native or invaded range). Given the similarity of the wasp pathogen fauna to that from honey bees, the lack of enemy release in wasp populations is probably related to spill-over or spill-back from bees and other social insects. Social insects appear to form a reservoir of generalist parasites and pathogens, which makes the management of wasp and bee disease difficult.

Alpha-lipoic acid differently affects the reserpine-induced oxidative stress in the striatum and prefrontal cortex of rat brain.

Antioxidative properties of alpha-lipoic acid (LA) are widely investigated in different in vivo and in vitro models. The aim of this study was to examine whether LA attenuates oxidative stress induced in rats by reserpine, a model substance frequently used to produce Parkinsonism in animals. Male Wistar rats were treated with reserpine (5 mg/kg) and LA (50 mg/kg) separately or in combination. The levels of reduced glutathione (GSH), glutathione disulfide (GSSG), nitric oxide (NO) and S-nitrosothiols as well as activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST) and L-gamma-glutamyl transpeptidase (gamma-GT) were determined in the striatum and prefrontal cortex homogenates. In the striatum and prefrontal cortex a single dose of reserpine significantly enhanced levels of GSSG and NO but not that of S-nitrosothiols when compared with control. In the striatum, LA administered jointly with reserpine markedly increased the concentration of GSH and decreased GSSG level. In the prefrontal cortex, such treatment produced only an increasing tendency in GSH level but caused no changes in GSSG content. In both structures LA injected jointly with reserpine markedly decreased NO concentrations but did not cause significant changes in S-nitrosothiol levels when compared with control. Enzymatic activities of GPx and GST were intensified by LA in the striatum. In the prefrontal cortex, GPx activity was not altered, while that of GST was decreased. Gamma-GT activity was attenuated by reserpine in the striatum while LA reversed this effect. Such changes were not observed in the prefrontal cortex. The mode of LA action in the striatum during the reserpine-evoked oxidative stress strongly suggests that this compound may be of therapeutic value in the treatment of Parkinson's disease.

Effect of acute administration of 1,2,3,4-tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of gamma-glutamyl transpeptidase in dopaminergic structures of rat brain.

The effect of acute administration of 1,2,3,4-tetrahydroisoquinoline, an endogenous substance suspected of producing Parkinsonism in humans, on the levels of glutathione and reactive oxygen species and on the enzymatic activity of gamma-glutamyl transpeptidase was investigated in the substantia nigra, striatum and cortex of rat brain. Four hours after a single dose of 1,2,3,4-tetrahydroisoquinoline (100 mg/kg i.p.), a significant increase in tissue glutathione level was found in the dopaminergic structures studied. The most pronounced effect was observed in the substantia nigra and cortex, and the weakest in the striatum. At the same time, significant inhibition of gamma-glutamyl transpeptidase was observed in the substantia nigra, cortex and striatum whose extent strictly corresponded to the increase in glutathione levels in those structures. Moreover, in 1,2,3,4-tetrahydroisoquinoline-treated rats, the production of reactive oxygen species was significantly reduced in the substantia nigra, whereas it was markedly enhanced in the striatum.Our results suggest that the increase in tissue glutathione level in the dopaminergic structures studied results from inhibition of gamma-glutamyl transpeptidase and refers to the extracellular pool of this peptide. Moreover, it is likely that both the 1,2,3,4-tetrahydroisoquinoline-induced alterations in glutathione level and the enhanced production of reactive oxygen species in the striatum may have implications for the pathogenesis of Parkinson's disease.

Effects of diallyl disulfide and other donors of sulfane sulfur on the proliferation of human hepatoma cell line (HepG2).

Effects of potential sulfane sulfur precursors (diallyl disulfide, cystamine, 2-mercaptoethanol disulfide, thiosulfate, immunothiole and pyridoxal phosphate jointly with cystine) on [3H]-thymidine incorporation in human hepatoma (HepG2) cells were studied. Of the tested compounds, diallyl disulfide, cystamine and 2-mercaptoethanol disulfide were found to cause significant inhibition of HepG2 cells proliferation. Moreover, pyridoxal phosphate jointly with cystine suppressed [3H]-thymidine incorporation, but the differences between that system and control cells were insignificant. In the case of thiosulfate, no significant difference was observed. The present study shows that diallyl disulfide, found in garlic, is effective in inhibiting [3H]-thymidine incorporation in human hepatoma HepG2 cell cultures. Similar antiproliferative effects on HepG2 cells are shown by such systems being a source of sulfane sulfur as cystamine or 2-mercaptoethanol disulfide. Thus, it may be concluded that these donors of reactive sulfane sulfur may be responsible for inhibition of the proliferation of HepG2 cells. It is suggested that the observed antiproliferative properties of the investigated compounds are connected with the presence of the highly reactive sulfane sulfur.

[Why does nitroglycerin tolerance appear?]. / Dlaczego pojawia sie tolerancja na nitrogliceryne?

Nitroglycerin (NTG) and other organic nitrates are beneficial drugs for the treatment of acute forms of coronary artery disease. The major limitation of organic nitrates use in clinical practice is the rapid development of hemodynamic tolerance. In general mechanisms of tolerance can be categorized into two different groups. One group of mechanisms is related to changes in the vascular smooth muscle bioactivation of organic nitrates. The other mechanisms are related to physiological response to increase nitric oxide (NO) production.

Biosynthesis and biological properties of compounds containing highly reactive, reduced sulfane sulfur.

The covalent modifications of sulfhydryl groups (-SH) may occur through oxidation to mixed disulfides (S-thiolation), S-nitrosylation, as well as persulfide and trisulfide formation. The latter possibilities of -SH group modification connected with compounds containing sulfur called sulfane sulfur are described in this paper. Sulfane sulfur compounds contain a labile, highly reactive sulfur atom at a reduced oxidation state with a valence of 0 or -1, covalently bound to another sulfur atom. These compounds include persulfides, polysulfides, polythionates, thiosulfate, elemental sulfur and disulfides, which enable tautomerization to thiosulfoxides. Sulfane sulfur compounds are formed in the anaerobic cysteine sulfur metabolism with the participation of such enzymes as cystathionase (CST), 3-mercaptopyruvate sulfurtransferase (MpST) and rhodanese (thiosulfate: cyanide sulfurtransferase). Compounds containing sulfane sulfur participate in cell regulation processes through activation or inactivation of some enzymes. Other important roles of sulfane sulfur compounds are their antioxidative properties, significance in the processes of carcinogenesis, participation in the tRNA sulfuration as well as an influence on the activity of immune cells. To recognize completely the biological role of compounds with sulfane sulfur it is necessary to have sensitive methods of quantitative determination, so a review of these methods is presented in this paper. Moreover, biosynthetic pathways and biological properties of these compounds have been discussed.

Effect of thiol drugs on the oxidative hemolysis in human erythrocytes.

The effect of different thiol drugs and 2-methyl-thiazolidine-2,4-dicarboxylic acid on the oxidative stress, induced by hydrogen peroxide, was examined in human erythrocytes. The results indicated that captopril (CA), methimazole, N-acetylcysteine (NAC), penicillamine and precursor of L-cysteine 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP) might protect the erythrocyte membrane against lipid peroxidation in the experimental conditions. Captopril, methimazole and penicillamine had the strongest antioxidative properties at the concentration level of 0.5 mM. The protective effects gradually decreased at higher and lower concentrations of these drugs. Contrary, the antioxidative properties of N-acetylcysteine increased with its levels growing in the reaction mixture, and only N-acetylpenicillamine did not protect erythrocytes against oxidative damages. The effect of 2-methyl-thiazolidine-2,4-dicarboxylic acid showed in these in vitro experimental conditions that it could act as an antioxidant at the concentration as high as 5 mM and higher.

The effect of nitrogen oxide level modulation on the content of thiol compounds and anaerobic sulfur metabolism in mice brains.

Aminoguanidine (AG) an inhibitor of NO-synthase reduces cysteine (Cys), cystine (CC), sulfane sulfur (SS) and glutathione (GSH) in brain stems but practically has no effects on the levels of reactive oxygen species (ROS). In cortex AG decreases to a lower degree the concentration of Cys, CC, GSH but in this brain part significantly decreases ROS levels and increases SS. Under the AG action cystathionase (CST) activity very seriously decreases in stems and in cortex and simultaneously activity of 3-mercaptopyruvate sulfurtransferase (MPST) increases. The morpholinosydnonimine (SIN-1) the specific donor of NO and O2 only slightly reduces Cys and GSH in brain stems and ROS and SS remain at the control levels. Simultaneously, there is an increase in cortex of the amounts of GSH with the reduction of ROS and SS. Furthermore, SIN-1 seriously decreases in stems and cortex the activity of CST and increases the activity of MPST. These results confirmed the relationship between intracellular levels of NO, sulfhydryl groups, ROS, and anaerobic sulfur metabolism.

Protective effect of alpha-keto acids on the oxidative hemolysis.

We studied antioxidative properties of various concentrations (0.3-2 mM) of biochemically important alpha-keto acids: pyruvate, alpha-ketoglutarate, oxaloacetate, glyoxylic acid as aldehyde acid and also 2-methyl-thiazolidyne-2,4-dicarboxylic acid and their effect on the oxidative hemolysis of human erythrocytes induced by hydrogen peroxide (H2O2). Normal erythrocytes proved to be very resistant to oxidative damages, so the high concentration of H2O2 (10 mM) as well as the presence of natrium azide, a catalase inhibitor, was necessary. The levels of malonyldialdehyde (MDA), reactive oxygen species (ROS) and hemoglobin (Hb) released were evaluated as the measure of red cell peroxidative hemolysis. Pyruvate, at the lowest used concentration (0.3 mM), caused the inhibition of lipid peroxidation (MDA) and a drop in the level of ROS, as well as a diminution of the degree of hemolysis and the effects were stronger than those of other alpha-keto acids. At the highest (2 mM) concentration, the protective effect against oxidative damage of all the investigated alpha-keto acids was similar and amounted to nearly 50% in relation to the control sample. On the contrary, in the case of aldehyde acid, e.g. glyoxylic acid, no protective effect in the same range of concentrations was found. This confirms the participation of non-enzymatic oxidative decarboxylation of alpha-keto acids in the hydrogen peroxide decomposition process.

2-Methyl-thiazolidine-2,4-dicarboxylic acid as prodrug of L-cysteine. Protection against paracetamol hepatotoxicity in mice.

Toxic doses of paracetamol (acetaminophen) destroy the cellular defense system in hepatic tissue. The degree of the destruction can be assessed be measuring the metabolism of sulfhydryl compounds, oxygen radicals and the release of certain enzymes. Administration of 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP; 1.2 mmol/kg) to mice 12 h prior to a toxic dose of paracetamol (600 mg/kg) suppressed the increase of aminotransferase activities in blood serum and the levels of reactive oxygen species in liver tissue. A protective effect of CP was also observed with respect to depletion of non-protein sulfhydryl compounds, cysteine and glycogen. The findings demonstrate that the cysteine prodrug CP is effective in preventing liver damage of a hepatotoxic dose of paracetamol in vivo. A further advantage of the new compound is the long duration of the effect of more than 12 h.

2-Methyl-thiazolidine-2,4-dicarboxylic acid protects against paracetamol induced toxicity in human liver derived HepG2 cells.

The effects of 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP) on paracetamol-induced toxicity were investigated and evaluated in a human liver derived HepG2 cell line. Incubation of the cells with CP (2 mM and 10 mM) drastically attenuated the GSH and cysteine depletion caused by toxic concentrations of paracetamol (1 mM and 5 mM). When CP (10 mM) was introduced alone into the medium, the level of malondialdehyde and the reactive oxygen species were maintained at the control levels with a simultaneous increase of non-protein sulfhydryl in the cells. Thus, the results of our work prove that CP is a non-toxic precursor of cysteine and GSH, and successfully prevents paracetamol toxicity in HepG2 cells.

Selective effect of 2-(polyhydroxyalkyl)-thiazolidine-4-carboxylic acids on nonprotein sulfhydryl groups in tumor bearing mice.

1. Thiazolidine derivatives (TD), the products of condensation of L-cysteine (cys) with sugars (D-glucose, D-xylose, D-arabinose, D-galactose, and D-mannose), successfully elevated nonprotein sulfhydryl (NPSH) levels in livers of Ehrlich ascites tumor (EAT)-bearing mice. 2. At the same time, TD promoted a significant drop of NPSH in EAT cells. 3. Thus, TD, through their selective influence on the levels of NPSH in liver and cancer cells appear to be promising compounds for anticancer therapy.

Sulfurtransferases activity and the level of low-molecular-weight thiols and sulfane sulfur compounds in cortex and brain stem of mouse.

The level of 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese activity and the level of sulfane sulfur compounds, L-cysteine and non-protein sulfhydryl groups (NPSH) were compared in cortex and brain stem of mouse. The level of cysteine and sulfane sulfur compounds was higher in brain stem; 107% and 217% of the value determined in cortex, respectively. The activity of MPST and rhodanese showed also increased level in brain stem; 114% and 119% of the value determined in cortex, respectively. The level of NPSH in cortex was about 10% higher in comparison to brain stem. It seems that in brain stem metabolism of sulfane sulfur compounds and/or their accumulation may occur in a higher degree than in cortex.

Selective modulation of non-protein sulfhydryl levels in Ehrlich ascites tumor bearing mice.

Thiazolidine derivatives (TD), prodrugs of L-cysteine (cys), synthesized by the condensation of cys with formaldehyde (thiazolidine-4-carboxylic acid-CF), acetaldehyde (2-methyl-thiazolidine-4-carboxylic acid-CA) and pyruvate (2-methylthiazolidine-2,4-dicarboxylic acid-CP), as well as amino acids thiocystine (T-cys) and cys, but not methionine (met) and S2O3(-2), successfully elevated non-protein sulfhydryl (NPSH) levels in livers of Ehrlich ascites tumor cells (EATC) bearing mice. At the same time, CA, promote a significant drop of NPSH concentration in EATC, whereas the other sulfur compounds (S-comp) have no effects. Thus TD and T-cys through their selective influence upon the level of NPSH in the liver and in cancer cells, seem to be the most interesting compounds for further studies on anticancer therapy.

The modulation of IL-2 dependent proliferation of CTLL-2 cells by 2-methyl-thiazolidine-2,4-dicarboxylic acid.

It is known that cysteine and other thiol compounds are able to modulate the immune response. The extracellular concentration of cysteine was shown to determine the intracellular level of glutathione (GSH). Thus cysteine, by enhancing GSH production, is able to affect some T-cell functions like IL-2 dependent cell proliferation and the generation of cytotoxic T cells. However, physiologically blood plasma cysteine is maintained at a very low concentration. The use of cysteine as a therapeutic compound in vivo is strongly limited due to its cytotoxicity. Recent studies demonstrate that N-acetyl-cysteine (NAC) as well as a variety of thiazolidine derivatives (TDs), which are the products of the reaction of L-cysteine with carbonyl compounds, could serve as a 'delivery' system for cysteine into the cell. In the present study, we have shown that 2-methyl-thiazolidine-2,4,-dicarboxylic acid (CP), the product of condensation of L-cysteine and pyruvate, strongly increases the proliferation of one particular cell line, IL-2 dependent CTLL-2 cells. We have also shown that this compound significantly increases the intracellular level of non-protein sulfhydryls (NPSH), but we did not find any correlation between NPSH levels and cell viability and proliferation. In contrast to CP, free cysteine showed its toxic properties by affecting cell viability of different cell lines and also by cancelling the influence of CP on the proliferation of CTLL cells.

Ethanol-induced changes in the content of thiol compounds and of lipid peroxidation in livers and brains from mice: protection by thiazolidine derivatives.

Treatment of mice with ethanol for 5 days resulted in a drop of total glutathione in the liver, possibly due to an ethanol-stimulated increased conversion into L-cysteine. The levels of L-cysteine and the rate of lipid peroxidation were above control levels. Similar but less pronounced changes were observed with brain tissue. The continuation of the treatment with ethanol led to an adaptation in both tissues as assessed at days 10 and 15. These findings suggest induction of enzymes involved in the defence mechanisms against lipid peroxidation. However, at day 23 of treatment the levels of total glutathione and L-cysteine were reduced in the liver whereas lipid peroxidation was increased. Thus, a state of impaired defence mechanisms occurred during prolonged treatment. Interestingly, the concentration of total glutathione was increased in the brain suggesting protective mechanisms in this organ and possibly a supply from other organs. No increase of lipid peroxidation levels in the brain was observed. The substitution of the deficit of thiol compounds is a major problem because neither L-cysteine nor glutathione can be utilized for different reasons. Therefore, we treated mice with thiazolidine derivatives which can be regarded as 'frozen' L-cysteine. Two days of treatment with 2-methyl-thiazolidine-2,4-dicarboxylic acid were sufficient to observe an increase of total glutathione and free L-cysteine levels and a decrease of lipid peroxidation in the liver. These findings demonstrate a new treatment for the tissue-damaging effects of chronic ethanol ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiazolidine derivatives as source of free L-cysteine in rat tissue.

The present study demonstrates that a variety of thiazolidine-4-(R)-carboxylic acids (TDs) which are the products of reactions of L-cysteine (cys) with carbonyl compounds could serve as a "delivery" system for cys to the cell. Liberation of the amino acid can occur enzymatically as well as non-enzymatically. The two possibilities have been proven by identification of representative compounds. The most specific substrate for mitochondrial enzymatic oxidation was thiazolidine-4-carboxylic acid (CF), the product of the reaction of cys with formaldehyde, and the least metabolized TD was 2-methyl-thiazolidine-4-carboxylic acid (CA), the product of the reaction of cys with acetaldehyde. TDs formed from cys and different sugars were not metabolized at all in mitochondria. N-Formyl-L-cysteine (NFC) the intermediate product of mitochondrial metabolism of CF was ascertained by 1H-NMR spectroscopy whereas N-acetyl-L-cysteine (NAC), the predicted metabolite of CA, was not detected, possibly due to a fast turnover. The further enzymatic hydrolysis of NFC as well as NAC to free cys was demonstrated to take place in the cytoplasm. Non-enzymatic hydrolysis of TDs depended on the chemical nature of the substituents in the thiazolidine (Th) ring. The most stable compound was unsubstituted Th and the least stable were CGlu(D) and CA. Following non-enzymatic ring opening and hydrolysis, CA was converted to methyl-djenkolic acid, which equilibrates with CA. We have identified this new compound by 1H-NMR spectroscopy. TDs may cause both a decrease and an increase in the levels of SH-groups in mitochondria. In the case of the stable CF, which is metabolized only enzymatically, an increase in the levels of SH-groups in mitochondria was observed. This suggests that enzymatic control of the breakdown of TDs prevents overflowing of the cell with thiol groups. The latter seems to be induced by high concentrations of those TDs which are hydrolysed non-enzymatically. This process leads finally to a decrease in free SH-groups by different mechanisms. The findings demonstrate two different mechanisms by which TDs can provide cys to the cells. The biological and pharmacological consequences are discussed.

The effect of 2-substituted thiazolidine-4(R)-carboxylic acids on non-protein sulphydryl levels and sulphurtransferase activities in mouse liver and brain.

2-Substituted thiazolidine-4(R)-carboxylic acids (TD) were found to increase the concentration of non-protein sulphydryls (NPSH) and the activity of rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) and 3-mercaptopyruvate sulphurtransferase (EC 2.8.1.2) in mouse liver. These properties suggest TDs are potentially hepatoprotective compounds. However TDs also cause depletion of NPSH in the mouse brain and this may be the reason for their toxic side effects on the central nervous system.

Transamination and transsulphuration of L-cysteine in Ehrlich ascites tumor cells and mouse liver. The nonenzymatic reaction of L-cysteine with pyruvate.

1. The activity of cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese is much lower in Ehrlich ascites tumor cells (EATC) than in mouse liver. 2. Contrary to mouse liver homogenate, no synthesis of sulphane sulphur-containing compounds from L-cysteine is observed in EATC homogenate. 3. 2-Methyl-thiazolidine-2,4-dicarboxylic acid (CP), 2-methyl-thiazolidine-4-carboxylic acid (CA) and thiazolidine-4-carboxylic acid (CF) can be used as sources of low molecular-weight thiol compounds both in EATC and mouse liver homogenate. 4. Pyruvate formed from phosphoenolpyruvate (PEP) in EATC homogenates reacts with L-cysteine (L-CYS) to CP.