Time-resolved analysis of amino acid stress identifies eIF2 phosphorylation as necessary to inhibit mTORC1 activity in liver

Amino acid availability is sensed by GCN2 (general control nonderepressible 2) and mechanistic target of rapamycin complex 1 (mTORC1), but how these two sensors coordinate their respective signal transduction events remains mysterious. In this study we utilized mouse genetic models to investigate the role of GCN2 in hepatic mTORC1 regulation upon amino acid stress induced by a single injection of asparaginase. We found that deletion of Gcn2 prevented hepatic phosphorylation of eukaryotic initiation factor 2α to asparaginase and instead unleashed mTORC1 activity. This change in intracellular signaling occurred within minutes and resulted in increased 5'-terminal oligopyrimidine mRNA translation instead of activating transcription factor 4 synthesis. Asparaginase also promoted hepatic mRNA levels of several genes which function as mTORC1 inhibitors, and these genes were blunted or blocked in the absence of Gcn2, but their timing could not explain the early discordant effects in mTORC1 signaling. Preconditioning mice with a chemical endoplasmic reticulum stress agent before amino acid stress rescued normal mTORC1 repression in the liver of Gcn2-/- mice but not in livers with both Gcn2 and the endoplasmic reticulum stress kinase, Perk, deleted. Furthermore, treating wildtype and Gcn2-/- mice with ISRIB, an inhibitor of PERK signaling, also failed to alter hepatic mTORC1 responses to asparaginase, although administration of ISRIB alone had an inhibitory GCN2-independent effect on mTORC1 activity. Taken together, the data show that activating transcription factor 4 is not required, but eukaryotic initiation factor 2α phosphorylation is necessary to prevent mTORC1 activation during amino acid stress

Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase

The anti-leukemic agent asparaginase activates the integrated stress response (ISR) kinase GCN2 and inhibits signaling via mechanistic target of rapamycin complex 1 (mTORC1). The study objective was to investigate the protective role of activating transcription factor 4 (ATF4) in controlling the hepatic transcriptome and mediating GCN2-mTORC1 signaling during asparaginase. We compared global gene expression patterns in livers from wildtype, Gcn2 -/-, and Atf4 -/- mice treated with asparaginase or excipient and further explored selected responses in livers from Atf4 +/- mice. Here, we show that ATF4 controls a hepatic gene expression profile that overlaps with GCN2 but is not required for downregulation of mTORC1 during asparaginase. Ingenuity pathway analysis indicates GCN2 independently influences inflammation-mediated hepatic processes whereas ATF4 uniquely associates with cholesterol metabolism and endoplasmic reticulum (ER) stress. Livers from Atf4 -/- or Atf4 +/- mice displayed an amplification of the amino acid response and ER stress response transcriptional signatures. In contrast, reduction in hepatic mTORC1 signaling was retained in Atf4 -/- mice treated with asparaginase

Influence of physical loading on behavioral reactions of rats in cerebral hypoperfusion

A number of clinical studies demonstrate that individual characteristics such as stress tolerance and cognitive ability can be considered not only as diagnostic variables but also as risk factors for severe cerebral hypoperfusion. The aim of the study was to identify the typological features of the effect of physical activity on the behavioral reactions of rats during cerebral hypoperfusion. Material and methods. Cerebral hypoperfusion was modeled by bilateral carotid artery ligation. The study involved 280 rats, divided into equal subgroups, according to sex, stress tolerance and learning capacity. Of these, 112 animals were subjected to daily short-term swimming as a rehabilitation model. Before the simulation, and at 6, 8, 14, 21, 28, 35, 60 and 90 days postoperatively, the animals were tested using a Morris water maze and an “open field” test. Results and discussion. The dynamics of stress resistance and learning ability are related to the periodicity of inflammatory, necrotic, circulatory and reparative remodeling in the model used. After 2-3 months of study, cerebral hypoperfusion causes stabilization of indices characterizing stress resistance and cognitive functions below control values. A decrease in stress resistance begins earlier from day 6 of the study, while the index characterizing cognitive functions first decreases significantly after 3 weeks of study. Factors reducing the damaging effects of cerebral hypoperfusion were female gender, high baseline stress tolerance and learning ability. When assessing learning ability under physical load, animals with high baseline cognitive development showed earlier recovery dynamics during urgent adaptation and a significant increase during long-term adaptation

Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice

Obesity increases risk for liver toxicity by the anti-leukemic agent asparaginase, but the mechanism is unknown. Asparaginase activates the integrated stress response (ISR) via sensing amino acid depletion by the eukaryotic initiation factor 2 (eIF2) kinase GCN2. The goal of this work was to discern the impact of obesity, alone versus alongside genetic disruption of the ISR, on mechanisms of liver protection during chronic asparaginase exposure in mice. Following diet-induced obesity, biochemical analysis of livers revealed that asparaginase provoked hepatic steatosis that coincided with activation of another eIF2 kinase PKR-like endoplasmic reticulum kinase (PERK), a major ISR transducer to ER stress. Genetic loss of Gcn2 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as Atf5 and Trib3 failed to increase. Instead, mechanistic target of rapamycin complex 1 (mTORC1) signal transduction was unleashed, and this coincided with liver dysfunction reflected by a failure to maintain hydrogen sulfide production or apolipoprotein B100 (ApoB100) expression. In contrast, obese mice lacking hepatic activating transcription factor 4 (Atf4) showed an exaggerated ISR and greater loss of endogenous hydrogen sulfide but normal inhibition of mTORC1 and maintenance of ApoB100 during asparaginase exposure. In both genetic mouse models, expression and phosphorylation of Sestrin2, an ATF4 gene target, was increased by asparaginase, suggesting mTORC1 inhibition during asparaginase exposure is not driven via eIF2-ATF4-Sestrin2. In conclusion, obesity promotes a maladaptive ISR during asparaginase exposure. GCN2 functions to repress mTORC1 activity and maintain ApoB100 protein levels independently of Atf4 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway

The Use of Computed Tomography in the Diagnosis of Jejunal Diverticulosis Complicated by Diverticulitis and Intestinal Bleeding

The jejunal diverticulosis is rare and accounts for approximately 0.5–1 % of the general population. Diagnosis of the pathology is difficult even in the presence of symptomatic complications (perforation, abscess, peritonitis, sepsis, intestinal obstruction and bleeding), therefore, it is often overlooked or delayed, which requires high awareness and vigilance of physicians. Emergency surgical interventions are performed due to the lack of clear clinical symptoms and reliable radiological data in an average of 25 % of patients with complications of diverticulosis. In the presented case, multislice computed tomography (MSCT) with intravenous contrasting revealed multiple diverticula of the jejunum with reactively altered walls, infiltration of the adjacent cellular tissue and small gas inclusions, indicating diverticulum microperforation. Esophagogastroduodenoscopy (EGD) was performed which confirmed the jejunum bleeding, and was followed by laparotomy which confirmed multiple jejunal diverticula with microperforation and bleeding. Thus, timely MSCT in combination with EGD allowed to avoid complications of jejunal diverticulitis

CT Diagnostics of Intestinal Obstruction Caused by Invagination Due to the Jejunal Wall Lipoma

A rare case of jejunal lipoma complicated by intestinal intussusception in a 71-year-old man is presented. Abdominal computed tomography revealed intestinal intussusception caused by the jejunal wall lipoma, allowed to timely make a diagnosis and perform surgical treatment

THE USE OF DIFFERENT DONOR MATERIALS IN KERATOPLASTY (LITERATURE REVIEW)

In numerous pathological processes of cornea the keratoplasty is often the only method of treatment. As different types of donor material are used in keratoplasty, this literature review is devoted to the application of preserved material and autotransplantation as the most available. The preservation methods of cornea allograft are divided into physical and chemical ones [Klement A.A., 1967]. According to the shelf-life all preservation methods are relatively divided into short and long termed. For the keratoplasty in some cases auto-tissues of a patient can be used. Autoconjuctiva has substantial advantages. Suhina L.A., Perekrestov M.B., Sukhina I.V. (2008) developed a therapeutic keratoplasty with autoscleral flap under conditions of traumatic injury of cornea. For reconstructive surgery amniotic membrane is applied, that is widely used recently in native and preserved form as a bio-cover for cornea, especially, in eye burns. Amnion tissue is an effective bio-stimulant of regenerative processes. After burns a special attention in the corneal epithelization is paid to limbus stem cells. In case of limbus-cellular insufficiency, stem cells, cultured on amniotic membrane, are used. Descemet’s membrane transplantation is the most modern technique of posterior corneal layer transplantation. This technique has become very important in corneal EED. Layer-by-layer keratoplasty with the «Alloplant» biomaterial is suggested as an effective method of treatment for deep trophic corneal ulcers. In penetrating states of cornea the optimization method of temporary filling technique using corneal and scleral tissues or silicone rubber to close penetrating defects during keratoplasty. Kasparov A.A., Aladinskaya I.V., Trufanov S.V. proposed in patients with vascularized corneal spots to sew during keratoplasty an umbilical part of amniotic membrane to the limbus region around keratotransplant. Different types of keratoplasty are presented above where different types of donor material are used. The correct choice can predetermine beneficial outcomes (optic, tectonic, therapeutic, cosmetic) of surgical treatment

Characterization of Functionalized Side-Chain Liquid CrystalMethacrylates Containing Nonmesogenic Units by DielectricSpectroscopy

[EN] The dielectric response of a series of side-chain liquid crystal copolymers, SCLCPs, the poly[6-(4'-methoxyazobenzene-4'-oxy)hexyl methacrylate]-co-poly[methyl methacrylate]s, MeOAzB/MMA copolymers, is presented in the frequency range f = 10(-2) to 107 Hz and over the temperature interval T = 150 to 120 degrees C. The relaxation spectra of these polymers have been studied in terms of the complex dielectric permittivity (epsilon' and epsilon '') and the dielectric loss tangent, tan(delta). The electric modulus, M*, has been also calculated. It is possible to distinguish two relaxations zones, one at low temperatures (including gamma and beta relaxations) and another at higher temperatures (including the alpha and beta 1 relaxations), all of them reported for liquid crystalline poly(methacrylate)s. The individual relaxations have been analyzed using Havriliak-Negami (HN) functions, and the effect of conductivity at high temperatures is subtracted. The thermal activation of the relaxations at low temperatures is studied using the Arrhenius equations as a function of copolymer composition, while the alpha and beta 1 relaxations are analyzed using Vogel-Tammann-Fulcher equations. The activation entropy has been also evaluated for all the relaxations through the Eyring equation. The temperature ranges, activation energies, and entropies of the relaxations at low temperatures (gamma and beta) are similar in the homopolymer and copolymers. However, the introduction of MMA units promotes variations in all the parameters related to the relaxations associated with the motions of the ester groups adjoining the polymer backbone. Specifically, a decrease is observed in the activation entropy values of the beta 1 relaxation, which suggests that the activation of the local motions of the side groups involves smaller cooperative regions in the copolymers with respect to the homopolymer. This fact may account for the extinction of the smectic behavior, together with the dilution of the anisotropic interactions between the mesogenic units on increasing MMA content. The study of this beta 1 relaxation can be then applied to anticipate the formation and stability of smectic phases in functionalized SCLCPs, by controlling the local mobility resulting in different mesogenic behavior.This paper is meant to be a tribute and recognition to the research activity of Professor Giulio Sarti. The authors would like to thank Dr. Victor Saenz de Juano for his advice during the discussion of the results and also the Spanish Ministry of Science and Innovation, through the Research Projects ENE2007-67584-C03, UPOVCE-3E-013, IT2009-0074, and ENE2011-28735-C02-01 and two FPI and FPU predoctoral grants, and the financial support of the Generalitat Valenciana, through the Grisolia and Forteza programs and the ACOMP/2011/189 program. UPV is also thanked for additional support through the PAID 05-09-4331 and PAID-06-11 programs.Martinez-Felipe, A.; Santonja Blasco, L.; Badia, J.; Imrie, C.; Ribes-Greus, A. (2013). Characterization of Functionalized Side-Chain Liquid CrystalMethacrylates Containing Nonmesogenic Units by DielectricSpectroscopy. Industrial and Engineering Chemistry Research. 52(26):8722-8731. https://doi.org/10.1021/ie3031339S87228731522