Ectopic expression of a grape aspartic protease gene, AP13, in Arabidopsis thaliana improves resistance to powdery mildew but increases susceptibility to Botrytis cinerea.

The grape aspartic protease gene, AP13 was previously reported to be responsive, in Chinese wild Vitis quinquangularis cv. 'Shang-24', to infection by Erysiphe necator, the causal agent of powdery mildew disease, as well as to treatment with salicylic acid in V. labrusca×V. vinifera cv. 'Kyoho'. In the current study, we evaluated the expression levels of AP13 in 'Shang-24' in response to salicylic acid (SA), methyl jasmonate (MeJA) and ethylene (ET) treatments, as well as to infection by the necrotrophic fungus, Botrytis cinerea, and the transcript levels of VqAP13 decreased after B. cinerea infection and MeJA treatment, but increased following ET and SA treatments. Transgenic Arabidopsis thaliana lines over-expressing VqAP13 under the control of a constitutive promoter showed enhanced resistance to powdery mildew and to the bacterium Pseudomonas syringae pv. tomato DC3000, and accumulated more callose than wild type plants, while the resistance of transgenic A. thaliana lines to B. cinerea inoculation was reduced. In addition, the expression profiles of various disease resistance- related genes in the transgenic A. thaliana lines following infection by different pathogens were compared to the equivalent profiles in the wild type plants. The results suggest that VqAP13 action promotes the SA dependent signal transduction pathway, but suppresses the JA signal transduction pathway.

Purification and properties of lipoxygenase from wheat seedlings infected by Fusarium graminearum and treated by salicylic acid.

Lipoxygenase from wheat seedlings in normal conditions, infected by Fusarium graminearum and treated by salicylic acid was isolated. The isolated enzyme was purified by the methods of salting-out (60% ammonium sulphate), dialysis, gel-filtration and ion-exchange chromatography. Specific activity of the purified enzyme was 8.0-12.5 ΔЕ234/mg of protein, degree of purification ­ 11.6-15.3 times. The enzyme yield was 18.3-27.9%. Molecular mass of lipoxygenase is 90 kDa, amino acid composition is distinguished by a high content of glutamic acid, proline, valine, isoleucine, leucine and low level of histidine, tyrosine, phenylalanine, threonine, tryptophan, cystein. Research of lipoxygenase substrate dependence indicated that the enzyme catalysed with the maximum velocity of the reaction of arachidonic acid oxidation at a substrate concentration of 4.5 mM at pH 7.2, the reaction of linoleic acid oxidation at a substrate concentration of 4.5 mM at pH 7.2 and the reaction of linolenic acid oxidation at a substrate concentration of 9.0 mM at pH 8.0. The change of wheat lipoxygenase activity depending on genotype resistance to Fusarium graminearum and millieu of germination was shown. One of the manifestations of the protective effect of salicylic acid is its ability to induce changes of lipoxygenase activity.

Perinatal deiodinase 2 expression in hepatocytes defines epigenetic susceptibility to liver steatosis and obesity.

Thyroid hormone binds to nuclear receptors and regulates gene transcription. Here we report that in mice, at around the first day of life, there is a transient surge in hepatocyte type 2 deiodinase (D2) that activates the prohormone thyroxine to the active hormone triiodothyronine, modifying the expression of ∼165 genes involved in broad aspects of hepatocyte function, including lipid metabolism. Hepatocyte-specific D2 inactivation (ALB-D2KO) is followed by a delay in neonatal expression of key lipid-related genes and a persistent reduction in peroxisome proliferator-activated receptor-γ expression. Notably, the absence of a neonatal D2 peak significantly modifies the baseline and long-term hepatic transcriptional response to a high-fat diet (HFD). Overall, changes in the expression of approximately 400 genes represent the HFD response in control animals toward the synthesis of fatty acids and triglycerides, whereas in ALB-D2KO animals, the response is limited to a very different set of only approximately 200 genes associated with reverse cholesterol transport and lipase activity. A whole genome methylation profile coupled to multiple analytical platforms indicate that 10-20% of these differences can be related to the presence of differentially methylated local regions mapped to sites of active/suppressed chromatin, thus qualifying as epigenetic modifications occurring as a result of neonatal D2 inactivation. The resulting phenotype of the adult ALB-D2KO mouse is dramatic, with greatly reduced susceptibility to diet-induced steatosis, hypertriglyceridemia, and obesity.

CYP2J2 overexpression protects against arrhythmia susceptibility in cardiac hypertrophy.

Maladaptive cardiac hypertrophy predisposes one to arrhythmia and sudden death. Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) promote anti-inflammatory and antiapoptotic mechanisms, and are involved in the regulation of cardiac Ca(2+)-, K(+)- and Na(+)-channels. To test the hypothesis that enhanced cardiac EET biosynthesis counteracts hypertrophy-induced electrical remodeling, male transgenic mice with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP2J2-TG) and wildtype littermates (WT) were subjected to chronic pressure overload (transverse aortic constriction, TAC) or ß-adrenergic stimulation (isoproterenol infusion, ISO). TAC caused progressive mortality that was higher in WT (42% over 8 weeks after TAC), compared to CYP2J2-TG mice (6%). In vivo electrophysiological studies, 4 weeks after TAC, revealed high ventricular tachyarrhythmia inducibility in WT (47% of the stimulation protocols), but not in CYP2J2-TG mice (0%). CYP2J2 overexpression also enhanced ventricular refractoriness and protected against TAC-induced QRS prolongation and delocalization of left ventricular connexin-43. ISO for 14 days induced high vulnerability for atrial fibrillation in WT mice (54%) that was reduced in CYP-TG mice (17%). CYP2J2 overexpression also protected against ISO-induced reduction of atrial refractoriness and development of atrial fibrosis. In contrast to these profound effects on electrical remodeling, CYP2J2 overexpression only moderately reduced TAC-induced cardiac hypertrophy and did not affect the hypertrophic response to ß-adrenergic stimulation. These results demonstrate that enhanced cardiac EET biosynthesis protects against electrical remodeling, ventricular tachyarrhythmia, and atrial fibrillation susceptibility during maladaptive cardiac hypertrophy.

Emerging roles of cadmium and heme oxygenase in type-2 diabetes and cancer susceptibility.

Many decades after an outbreak of severe cadmium poisoning, known as Itai-itai disease, cadmium continues to pose a significant threat to human health worldwide. This review provides an update on the effects of this environmental toxicant cadmium, observed in numerous populations despite modest exposure levels. In addition, it describes the current knowledge on the link between heme catabolism and glycolysis. It examines novel functions of heme oxygenase-2 (HO-2) that protect against type 2-diabetes and obesity, which have emerged from diabetic/obese phenotypes of the HO-2 knockout mouse model. Increased cancer susceptibility in type-2 diabetes has been noted in several large cohorts. This is a cause for concern, given the high prevalence of type-2 diabetes worldwide. A lifetime exposure to cadmium is associated with pre-diabetes, diabetes, and overall cancer mortality with sex-related differences in specific types of cancer. Liver and kidney are target organs for the toxic effects of cadmium. These two organs are central to the maintenance of blood glucose levels. Further, inhibition of gluconeogenesis is a known effect of heme, while cadmium has the propensity to alter heme catabolism. This raises the possibility that cadmium may mimic certain HO-2 deficiency conditions, resulting in diabetic symptoms. Intriguingly, evidence has emerged from a recent study to suggest the potential interaction and co-regulation of HO-2 with the key regulator of glycolysis: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4). HO-2 could thus be critical to a metabolic switch to cancer-prone cells because the enzyme PFKFB and glycolysis are metabolic requirements for cell proliferation and resistance to apoptosis.

Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1.

Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent interferon-ß production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.

Common polymorphisms in GSTM1, GSTT1, GSTP1, GSTA1 and susceptibility to colorectal cancer in the Central European population.

BACKGROUND: Central Europe presents with the highest incidence of sporadic colorectal cancer (CRC) worldwide. As sporadic CRC represents a typical multifactorial disease, it is characterized by intense interaction of the genetic background with the environment. Glutathione S-transferases could act as attractive susceptibility genes for CRC, as they are directly involved in conjugation between glutathione and chemotherapeutics, environmental pollutants and a wide spectrum of xenobiotics. METHODS: In this study, we investigated associations of polymorphisms in glutathione S-transferases (GSTs) genes, that is GSTA1, GSTT1, GSTM1 and GSTP1, with CRC in a total of 197 cases and 218 controls originating from the Czech Central European population. Polymorphisms were assessed by polymerase chain reaction/restriction fragment length polymorphism-based methods, allele-specific multiplex and allelic discrimination by real-time polymerase chain reaction. RESULTS: None of investigated polymorphisms showed any associations with CRC, with the exception of GSTP1; where the heterozygote genotype Ile105Val was associated with decreased risk of CRC (P = 0.043). CONCLUSIONS: The frequencies observed in our study are in accordance with those from other European Caucasian populations. Based on our studies, examined variability in GST genes is not a major determinant of CRC susceptibility in the Central European population.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria.

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in ß1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5(-/-) mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5(-/-) mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that ß1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

Mannose-binding lectin deficiency in a child with recurrent infections.

We describe an 11 month old girl with mannose-binding lectin deficiency who presented with recurrent infections. Her mother and brother also were affected. Mannose-binding deficiency is common, and we suggest that testing for it should be included in the evaluation of children with increased susceptibility to infection.

Paraoxonase: a multifaceted biomolecule.

BACKGROUND: Paraoxonase enzyme was first identified as a protective barrier against organophosphorus poisoning. After painstaking research spanning the last three decades, the knowledge about this enzyme has increased immensely. The present review attempts to elaborate the role of paraoxonase enzyme in normal physiology as well as provide an overview of the various disorders in which the enzyme may have a role in etiopathogenesis. METHODS: The literature was searched from the websites of the National Library of Medicine (http://www.ncbi.nlm.nih.gov/) and Pub Med Central, the U.S. National Library of Medicine's digital archive of life sciences journal literature. RESULTS: Paraoxonase acts as an important antioxidant enzyme against oxidative stress. The enzyme has been implicated in the pathogenesis of a number of disorders including cardiovascular disorders, cancers etc. CONCLUSIONS: A better understanding of the molecular mechanism of the enzyme along with the regulatory circuits will help us to utilize agonists to potentiate the anti oxidant actions of the enzyme.

The p110delta isoform of phosphatidylinositol 3-kinase controls susceptibility to Leishmania major by regulating expansion and tissue homing of regulatory T cells.

Resistance to Leishmania major and most intracellular pathogens is usually associated with a strong T cell-mediated immunity, particularly a CD4(+) Th1 response. Mice with an inactivating knock-in mutation in the p110delta isoform of PI3K (referred to as p110delta(D910A)) show severely impaired T cell responses. Because a strong T cell response is thought to mediate resistance to intracellular pathogens, we examined the outcome of L. major infection in p110delta(D910A) mice. Paradoxically, p110delta(D910A) mice on "resistant" and "susceptible" genetic backgrounds showed more robust resistance manifested as significantly reduced lesion size and accelerated parasite clearance. This enhanced resistance was associated with dramatically diminished immune responses, including impaired cell proliferation and effector cytokine (IFN-gamma and TNF) production. Interestingly, the ability of macrophages and dendritic cells from p110delta(D910A) mice to produce NO and destroy Leishmania parasites was similar to those of wild-type mice. We show that the enhanced resistance of p110delta(D910A) mice was due to impaired expansion and effector functions of regulatory T cells (Tregs). Adoptive transfer studies demonstrated that p110delta(D910A) mice lost their increased resistance when given enriched Tregs from wild-type mice. We suggest on the basis of these and further observations that the lack of this enzyme prominently affects Treg expansion and homing to infection sites, and that in the absence of Tregs, weak Th1 responses are capable of containing parasites and prevent pathology. We also suggest that temporary pharmacological inhibition of this enzyme may be a very effective form of treatment against cutaneous leishmaniasis.

Matrix metalloproteinase inhibition attenuates atrial remodeling and vulnerability to atrial fibrillation in a canine model of heart failure.

BACKGROUND: Atrial structural remodeling occurs in evolving heart failure (HF) and is an important substrate for the development of atrial fibrillation (AF). The matrix metalloproteinases (MMPs) play a role in extracellular remodeling, and recent studies have demonstrated increased atrial MMP activity in HF. Whether increased MMP activity directly contributes to atrial remodeling and AF in the setting of HF remains unclear. The current study examined the effects of MMP inhibition on atrial structural remodeling and AF vulnerability during HF progression. METHODS AND RESULTS: Three groups of dogs (n = 5 each)--control normal dogs (controls) and 10 dogs subjected to simultaneous atrioventricular pacing (SAVP) for 2 weeks to induce HF and randomly assigned to treatment with placebo (SAVP-placebo) or a MMP inhibitor PGE-7113313, a MMP-1-sparing MMP inhibitor, 6 mg/kg orally twice daily (SAVP-MMPi)--were studied. SAVP-MMPi dogs had less AF inducibility (percent of burst attempts leading to AF episodes: 1.7 +/- 2.9 seconds vs. 23+/-19 seconds, mean +/- SD, P < .05) and maintenance (AF duration: 253 [105 to 326] vs. 1932 [1296 to 2724] seconds, median [25th-75th quartile], P < .05) than SAVP-placebo dogs. The SAVP-MMPi dogs had significantly smaller increases in atrial myocyte cross sectional area, collagen area fraction, and MMP-9 activity relative to controls than SAVP-placebo. There were, however, no significant differences in the changes in chamber dimension and function in the left atrium. CONCLUSIONS: This unique finding of an attenuation of the vulnerability to AF in conjunction with reduced myocyte hypertrophy and fibrosis after MMP inhibition suggests that heightened MMP activity in the atria contributes to atrial structural remodeling and AF promotion during evolving HF.

DUSP6 (MKP3) null mice show enhanced ERK1/2 phosphorylation at baseline and increased myocyte proliferation in the heart affecting disease susceptibility.

The strength and duration of mitogen-activated protein kinase signaling is regulated through phosphorylation and dephosphorylation by dedicated dual-specificity kinases and phosphatases, respectively. Here we investigated the physiological role that extracellular signal-regulated kinases 1/2 (ERK1/2) dephosphorylation plays in vivo through targeted disruption of the gene encoding dual-specificity phosphatase 6 (Dusp6) in the mouse. Dusp6(-/-) mice, which were viable, fertile, and otherwise overtly normal, showed an increase in basal ERK1/2 phosphorylation in the heart, spleen, kidney, brain, and fibroblasts, but no change in ERK5, p38, or c-Jun N-terminal kinases activation. However, loss of Dusp6 did not increase or prolong ERK1/2 activation after stimulation, suggesting that its function is more dedicated to basal ERK1/2 signaling tone. In-depth analysis of the physiological effect associated with increased baseline ERK1/2 signaling was performed in cultured mouse embryonic fibroblasts (MEFs) and the heart. Interestingly, mice lacking Dusp6 had larger hearts at every age examined, which was associated with greater rates of myocyte proliferation during embryonic development and in the early postnatal period, resulting in cardiac hypercellularity. This increase in myocyte content in the heart was protective against decompensation and hypertrophic cardiomyopathy following long term pressure overload and myocardial infarction injury in adult mice. Dusp6(-/-) MEFs also showed reduced apoptosis rates compared with wild-type MEFs. These results demonstrate that ERK1/2 signaling is physiologically restrained by DUSP6 in coordinating cellular development and survival characteristics, directly impacting disease-responsiveness in adulthood.

Effective clearance of intracellular Leishmania major in vivo requires Pten in macrophages.

Leishmaniases are a major international public health problem, and macrophages are crucial for host resistance to this parasite. To determine if phosphatase and tensin homologue deleted on chromosome ten (Pten), a negative regulator of the PI3K pathway, plays a role in macrophage-mediated resistance to Leishmania, we generated C57BL/6 mice lacking Pten specifically in macrophages (LysMCrePten(flox/flox) mice). Examination of lesions resulting from Leishmania major infection showed that LysMCrePten(flox/flox) mice were more susceptible to the parasite than wild-type (WT) mice in the early phase of the infection, but were eventually able to eliminate the pathogen. In vitro Pten-deficient macrophages showed a reduced ability to kill parasites in response to IFN-gamma treatment, possibly because the mutant cells exhibited decreased TNF secretion that correlated with reductions in inducible nitric oxide synthase expression and nitric oxide production. In response to various TLR ligands, Pten-deficient macrophages produced less TNF and IL-12 but more IL-10 than WT cells. However, analysis of cells in the lymph nodes draining L. major inoculation sites indicated that both LysMCrePten(flox/flox) and WT mice developed normal Th1 responses following L. major infection, in line with the ability of LysMCrePten(flox/flox) mice to eventually eliminate the parasite. Our results indicate that the efficient clearance of intracellular parasites requires Pten in macrophages.

Cunning factor: macrophage migration inhibitory factor as a redox-regulated target.

Macrophage migration inhibitory factor (MIF) has an amazing history of rediscoveries and controversies surroundings its true biological function. It has been classified as a powerful cytokine capable of inducing tumour necrosis factor (TNF)-alpha, IL-1beta, IL-6, IL-8, PGE2 along with its ability to override glucocorticoid activity in relation to TNF-alpha release from monocytes. However, our recent study has failed to reproduce findings on MIF as a factor with cytokine-inducing properties but it has confirmed that MIF is capable of inducing glucocorticoid-counter regulating activity and amplifying LPS-driven cytokine responses. The aim of this review is to analyse the plethora of data surrounding MIF not just as a cytokine, but also as a hormone-like molecule, enzyme with atypical properties and as a thioredoxin-like protein to address fundamental questions about MIF functionality.

CHK2 kinase: cancer susceptibility and cancer therapy - two sides of the same coin?

In the past decade, CHK2 has emerged as an important multifunctional player in the DNA-damage response signalling pathway. Parallel studies of the human CHEK2 gene have also highlighted its role as a candidate multiorgan tumour susceptibility gene rather than a highly penetrant predisposition gene for Li-Fraumeni syndrome. As discussed here, our current understanding of CHK2 function in tumour cells, in both a biological and genetic context, suggests that targeted modulation of the active kinase or exploitation of its loss in tumours could prove to be effective anti-cancer strategies.

Lysosomal leakage and lack of adaptation of hepatoprotective enzyme contribute to enhanced susceptibility to ethanol-induced liver injury in female rats.

BACKGROUND: Women exhibit greater liver damage than men after chronic alcohol consumption. Similar findings are reported in animal models. Here, we determined whether differential liver injury occurred in male and female rats after feeding these animals liquid diets containing either ethanol or isocaloric dextrose with fish oil as the sole source of lipid. METHODS: Control and ethanol liquid diets containing fish oil were pair-fed to male and female rats for 8 weeks. Liver damage was evaluated by triglyceride accumulation, lipid peroxide formation, serum transaminases, histological evaluation, and the activities of selected lysosomal and hepatoprotective enzymes. RESULTS: Fatty liver was detected after ethanol feeding in both genders, but in female rats, triglyceride levels were 60% higher, lipid peroxides were 2-fold higher, and inflammatory cells were more evident than in males. A 2-fold elevation of cathepsin B in hepatic cytosol fractions, indicating lysosomal leakage, was detected in ethanol-fed female rats but no such elevation was observed in males. The basal activity of the hepatoprotective enzyme, betaine-homocysteine methyltransferase was 4-fold higher in livers of control male rats than females, and the enzyme activity was further elevated in ethanol-fed male rats but not in females. CONCLUSIONS: Thus, female rats given ethanol in a diet containing fish oil exhibited more severe liver damage than males. We propose that this difference results, in part, from a greater tendency by females to accumulate hepatic fat, thereby enhancing the potential for oxidative stress, which in turn leads to hepatic inflammation. In addition, our findings indicate that female rats have a higher susceptibility to liver damage because of a reduced capacity for hepatoprotection.

Effect of a thiol proteinase inhibitor, E-64-d, on susceptibility to infection with Staphylococcus aureus in Chediak-Higashi syndrome (beige) mice.

We previously reported that abnormally down-regulated protein kinase C (PKC) activity is responsible for the impaired cellular function of natural killer cells and polymorphonuclear cells (PMNs), and the giant granule formation in fibroblasts in the beige mouse, an animal model of Chediak-Higashi syndrome. Here, we examine the effect of oral or intraperitoneal administration of E-64-d, which protects PKC from calpain-mediated proteolysis, on the impaired cellular function in PMNs from beige mice. We found that oral administration of E-64-d (12.5 mg/kg body weight per day) for three consecutive days, significantly improved the abnormally increased concanavalin A (Con A) cap formation and the decreased lysosomal enzyme activity in beige PMNs. In addition, E-64-d significantly improved the delayed bactericidal activity against Staphylococcus aureus. In contrast, E-64-d at the same dose did not affect these cellular functions in PMNs from C57BL/6J mice. We confirmed that the abnormal down-regulation of PKC after Con A stimulation was eliminated in PMNs from E-64-d-treated beige PMNs. We then examined whether the administration of E-64-d to beige mice improved the susceptibility to experimental infection with S. aureus (2x10(8)/mouse). Both intraperitoneal and oral administration of E-64-d to beige mice resulted in a significant increase in survival, whereas E-64-d at the same dose did not alter the survival rate in normal mice. These results suggest that the administration of E-64-d may be effective against severe bacterial infection in Chediak-Higashi syndrome.