Altered expression of antioxidant enzymes and autophagic proteins in transglutaminase 2 knockout mice

""Background. Transglutaminases (TGs) are ubiquitous calcium-dependent acyl-transferases, catalysing post-translational protein modifications. Among the family members, TG2 (or "tissue" transglutaminase) acts as a multifunctional protein regulating cell processes, including autophagy [1,2]. . TG2 is highly expressed in the nervous tissue and reportedly involved in neurodegenerative disorders [3]. Indeed, the pathophysiology of these diseases includes insoluble aggregate formation, and covalent cross-linking of pathogenic proteins by TG2 has been suggested. Another hallmark of neurodegeneration is dysregulated autophagy, thus making the role played by TG2 in this cellular process especially relevant. Interestingly, conditions promoting TG2 activity, such as low GTP and high calcium levels, associated with oxidative stress, occur in neurodegeneration. . The present study aims to clarify the role of TG2 in redox balance and autophagy. To this purpose, the expression levels of antioxidant enzymes and pro-autophagic proteins were investigated in various brain regions and liver of TG2-\\\/- mice. . . Materials and methods. Expression of antioxidant enzymes, namely superoxide dismutase 1 and 2 (SOD1, SOD2), catalase (CAT) and glutathione peroxidase 1 (GPX1), and autophagic proteins (Beclin1, LC3 and AMBRA1) were evaluated by Western blotting (WB) and immunohistochemistry in selected brain areas (neocortex, hippocampus, brainstem, and cerebellum) and liver of 12-month-old TG2-\\\/- and wild-type mice.. . Results. WB and immunohistochemical data reveal altered expression patterns of antioxidant enzymes in both liver and brain tissues. Of particular interest is the statistically significant decrease of CAT and SOD2 in the cerebellum and hippocampus of TG2-\\\/- mice, while unchanged levels of these proteins are detected in the neocortex and brainstem. In the knockout liver, even more dramatic reduction of CAT and SOD2 expression is found, while SOD1 is intriguingly upregulated. Concerning the effect of TG2 deletion on autophagy, Beclin1 is down-regulated in the neocortex and hippocampus of knockout mice.. . Conclusions. Overall, our data on TG2-\\\/- mice support the involvement of the transamidating enzyme in controlling redox balance of different organs, and in regulating autophagic flux [1-3]. The specific decrease in the peroxisomal enzyme CAT and in the mitochondrial protein SOD2 emphasizes the role of these organelles in oxidative stress management and their interplay in cell metabolism. Moreover, region-based differences in the effect of TG2 deletion may reflect multiple functions related to the organ, tissue and cell type.. "

Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope

The proapoptotic activity of the transcription factor p53 critically depends on the phosphorylation of serine 46 (p53S46P). Here, we show that syncytia containing p53S46P could be detected in lymph node biopsies from human immunodeficiency virus (HIV)-1 carriers, in the brain of patients with HIV-1–associated dementia and in cocultures of HeLa expressing the HIV-1 envelope glycoprotein complex (Env) with HeLa cells expressing CD4. In this latter model, cell death was the result of a sequential process involving cell fusion, nuclear fusion (karyogamy), phosphorylation of serine 15 (p53S15P), later on serine 46 (p53S46P), and transcription of p53 target genes. Cytoplasmic p38 mitogen-activated protein kinase (MAPK) was found to undergo an activating phosphorylation (p38T180/Y182P [p38 with phosphorylated threonine 180 and tyrosine 182]) before karyogamy and to translocate into karyogamic nuclei. p38T180/Y182P colocalized and coimmunoprecipitated with p53S46P. Recombinant p38 phosphorylated recombinant p53 on serine 46 in vitro. Inhibition of p38 MAPK by pharmacological inhibitors, dominant-negative p38, or small interfering RNA, suppressed p53S46P (but not p53S15P), the expression of p53-inducible genes, the conformational activation of proapoptotic Bax and Bak, the release of cytochrome c from mitochondria, and consequent apoptosis. p38T180/Y182P was also detected in HIV-1–induced syncytia, in vivo, in patients' lymph nodes and brains. Dominant-negative MKK3 or MKK6 inhibited syncytial activation of p38, p53S46P, and apoptosis. Altogether, these findings indicate that p38 MAPK-mediated p53 phosphorylation constitutes a critical step of Env-induced apoptosis

In vitro Evaluation of Antiviral Efficacy of a Standardized Hydroalcoholic Extract of Poplar Type Propolis Against SARS-CoV-2

Except for specific vaccines and monoclonal antibodies, effective prophylactic or post-exposure therapeutic treatments are currently limited for COVID-19. Propolis, a honeybee's product, has been suggested as a potential candidate for treatment of COVID-19 for its immunomodulatory properties and for its powerful activity against various types of viruses, including common coronaviruses. However, direct evidence regarding the antiviral activities of this product still remains poorly documented. VERO E6 and CALU3 cell lines were infected with SARS-CoV-2 and cultured in the presence of 12.5 or 25 mu g/ml of a standardized Hydroalcoholic Extract acronym (sHEP) of Eurasian poplar type propolis and analyzed for viral RNA transcription, for cell damage by optical and electron microscopy, and for virus infectivity by viral titration at 2, 24, 48, and 72 h post-infection. The three main components of sHEP, caffeic acid phenethyl ester, galangin, and pinocembrin, were tested for the antiviral power, either alone or in combination. On both cell lines, sHEP showed significant effects mainly on CALU3 up to 48 h, i.e., some protection from cytopathic effects and consistent reduction of infected cell number, fewer viral particles inside cellular vesicles, reduction of viral titration in supernatants, dramatic drop of N gene negative sense RNA synthesis, and lower concentration of E gene RNA in cell extracts. Interestingly, pre-treatment of cells with sHEP before virus inoculation induced these same effects described previously and was not able to block virus entry. When used in combination, the three main constituents of sHEP showed antiviral activity at the same levels of sHEP. sHEP has a remarkable ability to hinder the replication of SARS-CoV-2, to limit new cycles of infection, and to protect host cells against the cytopathic effect, albeit with rather variable results. However, sHEP do not block the virus entry into the cells. The antiviral activity observed with the three main components of sHEP used in combination highlights that the mechanism underlying the antiviral activity of sHEP is probably the result of a synergistic effect. These data add further emphasis on the possible therapeutic role of this special honeybee's product as an adjuvant to official treatments of COVID-19 patients for its direct antiviral activity

The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

Cystic fibrosis (CF) is a rare autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is F508del-CFTR (ΔF) which leads the encoded ion channel towards misfolding and premature degradation. The disease is characterized by chronic bronchopulmonary obstruction, inflammation and airways colonization by bacteria, which are the major cause of morbidity and mortality. The STING pathway is the main signaling route activated in the presence of both self and pathogen DNA, leading to Type I Interferon (IFN I) production and the innate immune response. In this study, we show for the first time the relationship existing in CF between resistant and recurrent opportunistic infections by Pseudomonas aeruginosa and the innate immunity impairment. We demonstrate through ex vivo and in vivo experiments that the pathway is inadequately activated in ΔF condition and the use of direct STING agonists, as 2′,3′-cyclic GMP-AMP (2’, 3’ cGAMP), is able to restore the immune response against bacterial colonization. Indeed, upon treatment with the STING pathway agonists, we found a reduction of colony forming units (CFUs) consequent to IFN-β enhanced production in Pseudomonas aeruginosa infected bone marrow derived macrophages and lung tissues from mice affected by Cystic Fibrosis. Importantly, we also verified that the impairment detected in the primary PBMCs obtained from ΔF patients can be corrected by 2’, 3’ cGAMP. Our work indicates that the cGAS/STING pathway integrity is crucial in the Cystic Fibrosis response against pathogens and that the restoration of the pathway by 2’, 3’ cGAMP could be exploited as a possible new target for the symptomatic treatment of the disease

Fatal Takotsubo syndrome in critical COVID-19 related pneumonia

COVID-19 can involve several organs and systems, often with indirect and poorly clarified mechanisms. Different presentations of myocardial injury have been reported, with variable degrees of severity, often impacting on the prognosis of COVID-19 patients. The pathogenic mechanisms underlying cardiac damage in SARS-CoV-2 infection are under active investigation. We report the clinical and autopsy findings of a fatal case of Takotsubo Syndrome occurring in an 83-year-old patient with COVID-19 pneumonia. The patient was admitted to Emergency Department with dyspnea, fever and diarrhea. A naso-pharyngeal swab test for SARS-CoV-2 was positive. In the following week his conditions worsened, requiring intubation and deep sedation. While in the ICU, the patient suddenly showed ST segment elevation. Left ventricular angiography showed decreased with hypercontractile ventricular bases and mid-apical ballooning, consistent with diagnosis of Takotsubo syndrome (TTS). Shortly after the patient was pulseless. After extensive resuscitation maneuvers, the patient was declared dead. Autopsy revealed a subepicardial hematoma, in absence of myocardial rupture. On histology, the myocardium showed diffuse edema, multiple foci of contraction band necrosis in both ventricles and occasional coagulative necrosis of single cardiac myocytes. Abundant macrophages CD68+ were detected in the myocardial interstitium. The finding of diffuse contraction band necrosis supports the pathogenic role of increased catecholamine levels; the presence of a significant interstitial inflammatory infiltrate, made up by macrophages, remains of uncertain significance

Critical Involvement of the ATM-Dependent DNA Damage Response in the Apoptotic Demise of HIV-1-Elicited Syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection

Contact with HIV-1 envelope protein elicits release of ATP through pannexin-1 channels on target cells; by activating purinergic receptors and Pyk2 kinase in target cells, this extracellular ATP boosts HIV-1 infectivity

Effects of the plasticiser DEHP on lung of newborn rats: catalase immunocytochemistry and morphometric analysis

Experimental administration of di-(2-ethylexyl)-phthalate (DEHP), a plasticiser employed in the fabrication of polyvinyl chloride (PVC), causes increases in lipid metabolising enzymes along with marked peroxisomal proliferation. The effects are found in several mammalian tissues, of which the rodent liver is the most responsive target. Leakage of DEHP from PVC devices is favoured by high temperature and contact with lipid-containing biological fluids. Since preterm babies are currently ventilated through endotracheal PVC tubes, it seemed worthwhile to investigate DEHP effects on immature mammalian lung. In this research, female rats were fed with DEHP in the last week of pregnancy and after delivery, and lungs were excised from 2-day-old pups. At this age, in fact, rat lung histological features closely resemble those found in 24- to 36-week-old human fetuses. In treated animals, morphometric analysis of histological parameters revealed a dramatic decrease in the number of parenchymal airspaces, together with significant increases in their mean size. Moreover, cytochemical detection of the peroxisomal marker catalase revealed an increase in the number of type II pneumocytes. Our findings closely resemble abnormal histological features observed in autoptic lung specimens from children affected with chronic lung diseases