Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS.

Angiotensin-converting enzyme 2 (ACE2), one of the key enzymes of the renin-angiotensin system (RAS), plays an important role in SARS-CoV-2 infection by functioning as a virus receptor. Angiotensin peptides Ang I and Ang II, the substrates of ACE2, can modulate the binding of SARS-CoV-2 Spike protein to the ACE2 receptor. In the present work, we found that co incubation of HEK-ACE2 and Vero E6 cells with the SARS-CoV-2 Spike pseudovirus (PVP) resulted in stimulation of the virus entry at low and high micromolar concentrations of Ang I and Ang II, respectively. The potency of Ang I and Ang II stimulation of virus entry corresponds to their binding affinity to ACE2 catalytic pocket with 10 times higher efficiency of Ang II. The Ang II induced mild increase of PVP infectivity at 20 microM; while at 100 microM the increase (129.74+/-3.99 %) was highly significant (p<0.001). Since the angiotensin peptides act in HEK ACE2 cells without the involvement of angiotensin type I receptors, we hypothesize that there is a steric interaction between the catalytic pocket of the ACE2 enzyme and the SARS-CoV-2 S1 binding domain. Oversaturation of the ACE2 with their angiotensin substrate might result in increased binding and entry of the SARS-CoV-2. In addition, the analysis of angiotensin peptides metabolism showed decreased ACE2 and increased ACE activity upon SARS-CoV-2 action. These effects should be taken into consideration in COVID-19 patients suffering from comorbidities such as the over-activated renin-angiotensin system as a mechanism potentially influencing the SARS-CoV-2 invasion into recipient cells.

Angiotensinogen and angiotensin-converting enzyme mRNA decrease and AT1 receptor mRNA and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of GLUT4 and peroxisome proliferator-activated receptor (PPARγ).

Elevated adiposity is one of the accompanying features of increased age in humans and animals. Angiotensin II (Ang II) is considered as growth promoting peptide to be involved in hypertrophic enlargement of adipose tissue. However, systemic renin-angiotensin system (RAS) seems to decrease with increased age of rats. Local adipose tissue RAS might be independent of the systemic one. Therefore we performed a comprehensive study using rats with increased age from 9 to 26 weeks and evaluated angiotensinogen, angiotensin-converting enzyme (ACE) and AT(1) receptor mRNA in epididymal adipose tissue by RT-PCR. In addition, we determined AT(1) receptor protein by Western blotting and Ang II binding. These RAS parameters were correlated with expression of selected adiposity-dependent proteins such as leptin, adiponectin, insulin-dependent glucose transporter (GLUT4) and PPARgamma. Angiotensinogen and ACE expression decreased with increased age and adiposity. On the contrary, AT(1) receptor mRNA and protein was significantly elevated in 26-week-old rats though the Ang II binding was not different between 9 and 26-week-old animals. These results suggest dynamic adaptation of local adipose tissue RAS components to increased age and adiposity most likely by decreasing local Ang II formation which is thereafter compensated by increased expression of AT(1) receptor. However, this increase in AT(1) receptor mRNA and protein is not reflected in increased receptor binding. We believe that this complex regulation of adipose tissue RAS slows down the negative age and adiposity related changes in adipose tissue leptin, adiponectin, GLUT4 and PPARgamma.

Establishment of the cell line expressing human prion protein on PrP0/0 background.

In this work we described preparation of the novel cell lines expressing human prion protein on PrP0/0-background. Prepared cell lines originated from the Nagasaki mice (PrP0/0) and showed a fibroblast phenotype. The expression level of human prion protein in the developed cell lines was comparable to the physiological expression measured in GT1-7 cells. A great advantage of the prepared cell lines was their short doubling time that allowed obtaining of a large amount of cells for the proteomic experiments. Newly established cell lines open a broad spectrum of applications in the prion research. Besides the study of physiological function of the prion protein or its interactome, the new cell lines could be successfully employed as a unique tool for the better understanding of key events in the pathogenesis of prion diseases.

Post-translational modifications of tau protein.

Microtubule-associated protein tau is a phosphoprotein whose expression and phosphorylation is developmentally regulated. Whereas in adult mammalian brain several isoforms are produced from a single gene by alternative splicing, in fetal brain only a single isoform exists, corresponding to the smallest of the tau isoforms. Main physiological function of tau is the promotion of assembly and stabilization of microtubular network, which is essential for normal axonal transport of vesicles within the neuron. In human, tau protein undergoes several posttranslational modifications: such as phosphorylation, truncation, nitration, glycation, glycosylation, ubiquitination and polyaminations. When these modifications are disturbed, they play a serious role during the pathogenesis of Alzheimer's disease (AD). Hyperphosphorylation and truncation as the early events in AD pathogenesis, play significant role in the formation of neurofibrillary pathology. Phosphorylated tau has reduced capability in binding to microtubules and hyperphosphorylation together with truncation contributes to the formation of pathological tau filaments. This leads to destabilization of microtubular network and subsequent impairment of microtubule associated axonal transport. Since many data suggest that sporadic AD is the "disease of posttranslational modifications" of tau protein, more detailed investigation of tau protein modifications is urgently needed in order to understand pathogenesis of sporadic Alzheimer's disease (Fig. 1, Ref. 86).

The role of oxidative stress in the pathogenesis of Alzheimer's disease.

Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD) as a relevant marker of neuronal degeneration. However it plays an important role not only in the pathogenesis of neurodegenerative diseases but also in other critical disorders like heart diseases, carcinogenesis and others. Oxidative stress is also associated with normal aging. In this review we discuss a crucial question: to what extent oxidative stress may be a causative factor in pathogenesis of AD type of neurodegeneration. The results of several recent epidemiological studies appeared to be controversial at this point. It is believed that antioxidant therapies may have beneficial effects at least in delaying disease progression and appearance of AD specific clinical symptoms. Since there is no cure for AD recently, healthy life style and antioxidants enriched nutrition (or even antioxidant therapy) may provide an effective way of fighting against this deleterious disease (Ref. 102).

First confirmed sheep scrapie with A136R154Q171 genotype in Slovakia.

The first confirmed evidence of scrapie in Slovakia was demonstrated in one sheep of the autochthonous Merino breed from the southeastern part of the country. The reported scrapie was diagnosed during compulsory transmissible spongiform encephalitis (TSE) screening of sheep over 9 months of age assigned for consumption. The positive ewe was 5-year-old, which did not show any clinical signs of scrapie. The presence of the proteinase-resistant prion protein (PrP) in brain was proved independently by two laboratories using two different immunochemical screening systems, namely the Prionics Check (Western blot analysis) and Enfer TSE enzyme-linked immunosorbent assay (ELISA). In addition, the genotyping analysis of PrP gene demonstrated the presence of PrP genotype from the high risk group R4. The affected sheep was homozygous for the allele PrP(ARQ) (ARQ/ARQ) coding for alanine (A), arginine (R) and glutamine (Q) at three most relevant codons (136, 154 and 171, respectively). The healthy sister of the positive ewe was heterozygous in the PrP locus and carried alleles ARQ/ARR.

Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

Thyroid hormone receptor occupancy and biological effects of 3,5,3,-L-triiodothyronine (T3) in GH4C1 rat pituitary tumour cells.

The GH4C1 pituitary cell line, an excellent model for a thyroid hormone action study, was used for determination of the relationship between thyroid hormone receptor occupancy and intensity of cell proliferation, prolactin (PRL) production, thyrotropin (TSH) inhibition and 3,5,3,-L-triiodothyronine (T3) receptor down-regulation. Nuclear receptor population was progressively occupied by T3 in concentrations ranging from 0.025 to 10.0 nM T3. Bmax ranged from 0.029 fmol/10(6) cells at the lowest T3 concentration to Bmax = 12.51 fmol/10(6) cells at the highest concentration. Each of the observed biological events is operative within distinct dose-response ranges in cultured GH4C1 cells. The maximal biological response (except the TSH inhibition and T3 receptor down-regulation) does not require the occupation of the whole nuclear receptor population by T3 and the intensity of none of the responses studied was directly proportional to thyroid hormone receptor occupancy.

Nuclear all-trans retinoic acid receptors: in vitro effects of selenium.

The present study was undertaken to investigate the effects of selenite (SeIV) and selenate (SeVI) on the all-trans retinoic acid (RA)-nuclear retinoic acid receptor (RAR) complex formation in rat liver. We also present the data on the in vitro effects of SeIV on the RARalpha and the type I iodothyronine 5'-deiodinase gene expression in the GH4C1 rat pituitary tumor cells. SeIV at 1.0 micromol/L was found to reduce (p < 0.05) the RA specific binding to RAR in rat liver. Dithiothreitol (DTT), a protective agent for sulfhydryl groups, was found to be slightly effective in protecting the RAR binding properties when affected by SeIV. SeVI at 0.1 micromol/L reduced (p < 0.05) the RA specific binding to RAR in liver, as well. Seleno-L-methionine (Se-II) when compared to L-methionine did not exert any inhibitory effect on the formation of the RA-RAR complex. SeIV (up to 2.5 micromol/L) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. SeIV at 1.0 micromol/L significantly decreases the rate of mRNA synthesis and/or degradation of the alpha form of the RAR and causes the enhancement of the type I iodothyronine 5'-deiodinase gene expression in GH4C1 cells. The results based on in vitro experiments suggest that inorganic selenium may affect the RA specific binding to their cognate receptor molecules, and it may reduce expression of the gene encoding the RARalpha, with the cell vitality and the cell growth remaining unchanged.

Regulation of the mouse cellular retinoic acid-binding protein-I gene by thyroid hormone and retinoids in transgenic mouse embryos and P19 cells.

The regulation of mouse cellular retinoic acid-binding protein-I (CRABP-I) gene expression by the retinoids and thyroid hormones was examined, by using a beta-galactosidase (lacZ) reporter gene and a CRABP-I specific antibody, in transgenic mouse embryos and a mouse embryonal carcinoma cell line P19. The CRABP-lacZ reporter gene expression recapitulated the expression pattern of endogenous CRABP-I in the developing central nervous system. In mid-gestation mouse embryos the expression of both the transgene and the endogenous protein was elevated under the condition of hypovitaminosis A, suggesting that depletion of retinoic acid (RA) induced CRABP-I expression in embryos. Consistently, this reporter was suppressed by RA in P19 cells. In co-transfection experiments it was demonstrated that the expression of RAR beta, RAR gamma or RXR alpha suppressed this reporter expression. In experiments designed to alter the thyroid hormone status in animals it was demonstrated that both the reporter gene and the endogenous CRABP-I expression were reduced by triiodothyronine injection and were elevated in a hypothyroidic condition induced by feeding with iodine-deficient diet supplemented with 6-propyl-2-thiouracil. In co-transfection experiments it was also demonstrated that the expression of T3R beta suppressed the reporter expression in P19 cells. It was concluded that RA had a suppressive effect on CRABP-I gene expression in embryos and P19 cells and the effect could be mediated through RAR beta, RAR gamma or RXR alpha. A role of thyroid hormones in CRABP-I gene expression and vitamin A metabolism in animals is discussed.

Both iso- and hyperosmotic ethanol stimulate release of hypothalamic thyrotropin-releasing hormone despite opposite effect on neuron volume.

Previous studies have indicated that isosmolar, but not hyperosmolar, ethanol induces in vitro gonadotropin-releasing hormone secretion from the basal hypothalamus, presumably by causing cell swelling. Moreover, ethanol reduces secretion of another hypothalamic neuropeptide vasopressin. We have studied the acute effect of ethanol on specific hypophysiotropic basal and K+-stimulated thyrotropin-releasing hormone secretion in vitro especially in relation to cell swelling. Isosmotic 40-160 mM ethanol increased thyrotropin-releasing hormone release from the hypothalamic paraventricular nucleus and median eminence in a dose-dependent manner. Both a 30% decrease of osmolarity and isosmotic 80 mM ethanol induced 12% swelling of hypothalamic neurons. Hyperosmotic 80 mM or 160 mM ethanol induced release of thyrotropin-releasing hormone from both hypothalamic structures but did not cause cell swelling (80 mM) or even induced cell shrinkage (160 mM). Depletion of medium Ca2+ did not affect thyrotropin-releasing hormone secretion caused by either isosmotic or hyperosmotic ethanol. Our data indicate that both iso- and hyperosmotic ethanol stimulated release of hypophysiotropic thyrotropin-releasing hormone despite opposite effects on neuron volume. The mechanism of ethanol action appears complex and variable depending on the type of cell and neuropeptide affected.

[The basis for the variable effects of thyroid hormones]. / Podstata variability úcinku hormónov stítnej zl'azy.

Among physiological reactions of the organism, the multiplicity of action of thyroid hormones is a well known phenomenon. Thyroid hormones (TH) regulate activity of other hormones, receptors for bioactive signals, enzymes, and the function of ion channels. Until triiodothyronine has been identified as a ligand for oncoprotein c-erbA, this broad potency of actions has been mysterious. C-erbA has been recognized to be a transcription factor and an authentic thyroid hormone receptor. Important evidence comes from molecular studies, which have described isoforms of c-erbA and other members of intranuclear receptor family, their heterodimerization, thyroid hormone responsive elements in the target genes, and the existence of the cross-talk mechanism. Till present, much has been learned about TH transcription regulations in vitro. An open question remains whether this regulations are operative also in the intact organism.

In vitro effects of sodium selenite on nuclear 3,5,3'-triiodothyronine (T3) receptor gene expression in rat pituitary GH4C1 cells.

The present study was undertaken in order to investigate the effects of sodium selenite on: 1. The growth of rat pituitary GH4C1 cells; 2. The nuclear T3 receptor gene expression; 3. The cytoplasmic protein phosphorylation; and 4. The prolactin secretion in rat pituitary GH4C1 cell line. Sodium selenite (up to 2.5 microM) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. On the other hand, 0.5 microM sodium selenite significantly decreases the rate of mRNA synthesis and/or degradation of both, the alpha 1 form of the T3 receptor (TR alpha 1) and the alpha 2 isoform of the T3 receptor. At 1 microM of sodium selenite, significant changes in the electrophoretic profile of low molecular mass cytoplasmic proteins were found, moreover, sodium selenite (1 microM) also considerably affects phosphorylation of a higher molecular mass proteins. The results based on the in vitro experiments suggest that sodium selenite may affect specific processes at the pretranslational level as well as it may also take part in processes of posttranslational modification of protein(s), the cell vitality and the cell growth remaining unchanged.

3,5,3'-L-triiodothyronine promotes survival and axon elongation of embryonic rat septal neurons.

The effect of 3,5,3'-L-triiodothyronine (T3) on survival and morphology of primary cultured neurons of the fetal rat brain was studied. In defined conditions of serum-free culture media we found the death preventing effect of T3 in all tested neuronal populations cultivated at high initial densities of plating (10(5) cells/cm2). While the survival of cerebrocortical neurons was improved very slightly, the number of surviving hippocampal and septal neurons reached 127.2 +/- 2.0% or 134.8 +/- 12.3% of their respective controls. The septal neurons responded at normal physiological concentration of T3 (1 nM) in high density as well as in low density cultures (5 x 10(3) cells/cm2). Moreover the treatment with 10 nM of T3 caused significant extension of the axon elongation of septal neurons (194.5 +/- 15.7%). These findings suggest the direct positive effect of T3 on pure cell population of septal neurons derived from embryonic rat brain and support the evidence for the role of this peripheral hormone during neuritogenesis.

Effect of selenite and selenate on rat liver nuclear 3,5,3'-triiodothyronine (T3) receptor.

The present study was undertaken to investigate the effects of selenite (SeIV) or selenate (SeVI) on nuclear T3 receptors of rat liver. Selenite at 0.1 microM (p < 0.01) inhibited the T3 specific binding to rat liver nuclear receptors. The specific binding of the T3 receptor was fully restored when even 1.0 microM selenite was separated from the T3 receptor by gel filtration. No inhibitory effect of selenite (up to 100 microM) on the T3 binding to nuclear receptor was found in the presence of 1.0 mM dithiothreitol. The rate of dissociation of the T3-nuclear receptor complex was effectively increased by 0.1 microM selenite. Selenate up to 1 mM as well as sulfite or sulfate up to 0.1 mM did not exert an inhibitory effect on T3 receptors. The results based on the in vitro experiments suggest that the selenium in the form of selenite may reversibly affect the T3 binding on the receptor molecule.

Effect of protease inhibitors and substrates on 3,5,3'-triiodothyronine binding to rat liver nuclear receptors.

The effect of protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-carbobenzoxy-L-phenylalanine chloromethyl ketone (ZPCK) at concentrations ranging from 1.5 x 10(-6) mol/l to 1.5 x 10(-4) mol/l on the specific binding of 3,5,3'-triiodothyronine (T3) to rat liver nuclear receptors was evaluated. Both TPCK at 1.5 x 10(-5) mol/l (P < 0.001) and ZPCK at 1.5 x 10(-6) mol/l (P < 0.05) showed the inhibition of T3 binding to nuclear receptors. The evaluation of T3 binding data following separation of unbound TPCK from treated nuclear receptors on a Sephadex G-25 column showed that the inhibition of T3 binding was irreversible, since a substantial decrease of the equilibrium association constant (Ka) was found when compared to the mock samples lacking TPCK. In addition, similar inhibitory effect on T3 specific binding to rat liver nuclear receptors was found at 5.0 x 10(-4) mol/l by protease substrates L-tyrosine benzyl ester (P < 0.001), L-tyrosine ethyl ester (P < 0.02), L-tryptophan methyl ester (P < 0.02) and L-tryptophan ethyl ester (P < 0.05). The data suggest that: 1. both protease inhibitors and substrates tested inhibited T3 specific binding to T3 nuclear receptors; 2. this may support the hypothesis that T3 receptors like other receptors encoded by c-erbA gene possess a site that recognize both serine protease inhibitors and substrates in its hormone binding domain.

Effect of triiodothyronine on rat liver polysome profiles and translational activity of mRNA after partial hepatectomy.

The administration of 3,5,3'-L-triiodothyronine (T3) (200 micrograms/100 g) to rats immediately after partial hepatectomy led to an increase in total polysome content of residual hepatocytes (as measured 18 h postoperatively) which exceeded that observed after partial hepatectomy in the absence of hormone. The percentage of free ribosome pool remained the same after partial hepatectomy irrespectively on the absence or presence of T3. After partial hepatectomy the sedimentation pattern of total polysomal fraction (isolated 18 h postoperatively) shifted towards smaller aggregates as compared with that isolated from sham operated animals. T3 did not affect this pattern. The translational capacity of mRNA isolated from regenerating liver after 18 h was higher than that of mRNA isolated from sham operated animals. T3 increased the translational capacity of mRNA isolated from regenerating liver if administered immediately after surgery. Two dimensional electrophoresis of translated products showed that one of the typical proteins recently characterized with Mr 25,000 pI 6.9 appeared after partial hepatectomy and was significantly increased when T3 was administered immediately after surgery. These results show that thyroid hormone can enhance some of the characteristics features of liver regeneration involved in the synthesis of proteins typical for the process of liver regeneration.

Thyroid hormone responsiveness of the L1210 murine leukemia cell line.

The presence of saturable and high affinity 3,5,3'-triiodothyronine (T3) binding sites was demonstrated in L1210 murine leukemia cell nuclei. Scatchard analysis revealed one class of receptors for T3 with Ka = 2.187 x 10(9) l/mol and a maximum binding capacity (Bmax) of 3.96 fmol/10(6) cells. The effects of T3 on protein phosphorylation and growth rate of L1210 cells were investigated in a medium containing T3-depleted fetal calf serum. T3 was observed to be effective in enhancing protein phosphorylation (153.06% +/- 5.99 SD) compared to cells grown in the absence of T3 (81.49% +/- 13.50 SD). Moreover, in the presence of high T3 concentration (11.15 nmol/l) T3 was found to significantly increase the cell growth rate. In addition, the T3 receptor-associated alterations during the cell cycle, as measured by flow cytometry, suggest that the presence of T3 receptors becomes evident during the late G1 phase of the cell cycle, and T3 receptor numbers increase during the S phase. These results suggest that in in vitro conditions representing high T3 concentration, the number of L1210 leukemia cells may be increased by T3 via nuclear receptors. The L1210 leukemia cell line may serve as a convenient tool for in vitro studies of nuclear receptors and/or mechanism of action of T3. The binding affinity of T3 receptors is similar to that found in rat hepatocytes or human lymphocytes.

Distribution of triiodothyronine nuclear receptors during the cell cycle in mouse leukemia cells.

The process of the appearance of thyroid hormone receptors in the cell cycle was studied in mouse leukemia cells L1210. After cell synchronization by 2 mM thymidine for 12 h followed by 80 nM Colcemid for 4 h, the specific binding of triiodothyronine (T3) to its nuclear receptors was determined 3, 6, 9 and 12 h after release from the thymidine-Colcemid block. Three h after release from the block, [125I]T3 specific binding was 11.4 +/- 2.5% of a control value measured for an asynchronous population. An upward slope in progression of T3 nuclear receptors was found 6 h (32.3% +/- 4.5%), 9 h (47.8% +/- 5.2%) and 12 h (83% +/- 4.3%) after release from thymidine-Colcemid block. The data suggest that processes involving T3 receptor promotion in cell nuclei are operative within the late G1 and S phases of the cell cycle, and thus the increase in T3 receptor concentration in the nuclei is in a positive correlation with the number of cells in the S compartment of the cell cycle.