The hypoxia-regulated transcription factor DEC1 (Stra13, SHARP-2) and its expression in human tissues and tumours

DEC1, also known as SHARP-2 or Stra13, is an important molecule in embryonic differentiation and has recently been identified to be strongly inducible by hypoxia. Its distribution in normal human tissues and most tumour types is unknown. In the present study, a polyclonal antiserum to a 10-amino acid peptide from DEC1 has been raised. Using this antiserum, DEC1 was shown to be widely expressed in most normal human tissues, but usually only in a proportion of cells and typically with a nuclear localization. In tumours, expression was either augmented (the commonest pattern) or occasionally decreased. Similarly, in most normal tissues, low or absent expression was observed in endothelial cells, whereas in many tumour samples endothelium was usually strongly positive. In tumours, there was a striking pattern of staining seen in connection with areas of necrosis, with absence of DEC1 expression within a zone of morphologically viable cells immediately adjacent to the necrotic zone. This suggests that while DEC1 may be up-regulated by hypoxia in cancer, in more extreme hypoxia it may have a role in cell death. Its interrelationship with other hypoxically regulated molecules, such as the hypoxia-inducible factors or carbonic anhydrase IX, and differentiation of tumours now requires further investigation.     

In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes

Binding of apoptotic cells was compared after incubation of thymocytes with two clones of murine thymic stromal cells to which CD4(+)/CD8(+) thymocytes attach. With the BA/10, but not the BA/2, clone, thymocytes with apoptotic morphology were bound irreversibly. These tightly bound thymocytes were further identified as apoptotic in terms of active caspase-3 and DNA fragmentation assayed in situ. FACS analysis indicated that the apoptotic thymocytes are at an early double-positive stage and results with mice mutant for the Fas gene showed that the Fas-Fas ligand system is not involved. Comparison of BA/10 and BA/2 cells showed that the former, but not the latter, can be induced to express CDR-1 antigen which is characteristic of cortical epithelial thymic stroma and constitutively express DEC-205, a surface protein common to cortical thymic epithelium and dendritic cells. Antibody NLDC-145 that is specific for the DEC-205 protein strongly reduced the number of stromal cells with bound apoptotic thymocytes. Preincubation of thymocytes in dexamethasone dramatically increased the number of bound apoptotic cells, indicating that the thymic cortical epithelial cells can participate in clearance of apoptotic thymocytes through involvement of DEC-205.     

5-Hydroxytryptamine4 receptors mediate relaxation of the rat oesophageal tunica muscularis mucosae

Summary The present study was designed to characterize an atypical 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the rat oesophageal tunica muscularis mucosae. All experiments were performed under equilibrium conditions, using pargyline to inhibit the oxidative deamination of indoleamines, and cocaine and corticosterone to inhibit neuronal and extraneuronal uptake. Under these conditions 5-HT (0.3–1000 nmol/l) produced a concentration-dependent relaxation of carbachol-induced tension. The concentration-effect curve to 5-HT was unaffected by potent antagonists for 5-HT₁, 5-HT₂, 5-HT₃ and so called 5-HT_1P receptors (metergoline, methysergide, ketanserin, ondansetron, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide), but was antagonized competitively by ICS 205–930 (pA₂ = 6.7). Responses to 5-HT were mimicked by other indoleamines and substituted benzamides with the following order of potency: 5-HT 5-methoxytryptamine > cisapride = -methyl-5-HT = (S)-zacopride = renzapride > (RS)-zacopride > 5-carboxamido-tryptamine = metoclopramide = (R)-zacopride > tryptamine > 2-methyl-5-HT. ICS 205–930 afforded similar pA₂ values (6.0–6.7) against each agonist, indicating a common site of action. Concentration-effect curves to 5-HT were not affected by tetrodotoxin or indomethacin, sugesting that 5-HT-induced relaxation of the tunica muscularis mucosae was mediated via a postjunctional receptor, independent of endogenous prostanoids. The pharmacological profile of the 5-HT receptor in the rat oesophageal tunica muscularis mucosae correlates well with the 5-HT₄ receptor characterized recently in both the CNS and gastro-intestinal tract. Send offprint requests to D. E. Clarke at the above address     

Noradrenergic and dopaminergic interactions in escape behavior: Analysis of uncontrollable stress effects

The effects of norepinephrine receptor blockade on the deficits of escape behavior induced by haloperidol and by inescapable shock were evaluated. Phenoxybenzamine, the -norepinephrine receptor blocker, was found to enhance escape behavior and to eliminate the disruptive effects of both inescapable shock and haloperidol. In contrast, the -norepinephrine receptor antagonist, propranolol, was without effect on behavior under any of these conditions, while the dopamine--hydroxylase inhibitor, FLA-63, disrupted performance. Like phenoxybenzamine, the norepinephrine receptor stimulant, clonidine, was found to eliminate the behavioral disruption produced by haloperidol. These somewhat paradoxical findings were discussed in terms of the contribution of DA-NE interactions in determining behavioral change in aversive paradigms.     

Dynamic mislocalizations of nuclear pore complex proteins after focal cerebral ischemia in rat

Nuclear pore complexes (NPCs) play an important role in coordinating the transport of proteins and nucleic acids between the nucleus and cytoplasm, and are therefore essential for maintaining normal cellular function and liability. In the present study, we investigated the temporal immunohistochemical distribution of five representative components of NPCs—Ran GTPase‐activating protein 1 (RanGap1), glycoprotein‐210 (Gp210), nucleoporin 205 (Nup205), nucleoporin 107 (Nup107), and nucleoporin 50 (Nup50)—after 90 min of transient middle cerebral artery occlusion (tMCAO) up to 28 days after the reperfusion in rat brains. Single immunohistochemical analyses showed ring‐like stainings along the periphery of the nucleus in sham control brains. After tMCAO, Gp210 and Nup107 immunoreactivity continuously increased from 1 day, and RanGap1, Nup205, and Nup50 increased from 2 days until 28 days, which also displayed progressive precipitations within the nucleus in the peri‐ischemic area, while the ischemic core showed scarce expression with collapsed structure. Double immunofluorescent analyses revealed nuclear retention and apparent colocalization of RanGap1 with Nup205, Gp210 with Nup205, and partial colocalization of Nup205 with Nup107; most of the ischemic changes above were similar to those observed in patients with C9orf72‐genetic amyotrophic lateral sclerosis. Taken together, these observations suggest that the mislocalization of these nucleoporins may be a common pathogenesis of both ischemic and neurodegenerative disease. © 2016 Wiley Periodicals, Inc.     

Evaluation of myocardial performance index in patients with COVID-19: An echocardiographic follow-up study

Introduction and ObjectivesMyocardial performance may be impaired in cytokine-mediated immune reactions. The myocardial performance index (MPI) is a practical parameter that reflects systolic and diastolic cardiac function. We aimed to assess the MPI in patients with COVID-19.MethodsThe study population consisted of 40 healthy controls and 40 patients diagnosed with COVID-19 who had mild pneumonia and did not need intensive care treatment. All participants underwent echocardiographic examination. First, the MPI and laboratory parameters were compared between healthy controls and patients in the acute period of infection. Second, the MPI and laboratory parameters were compared between the acute infection period and after clinical recovery.ResultsCompared with healthy controls, patients with COVID-19 had a significantly higher MPI (0.56±0.09 vs. 0.41±0.06, p<0.001), longer isovolumic relaxation time (IRT) (112.3±13.4 vs. 90.6±11.2 ms, p<0.001), longer deceleration time (DT) (182.1±30.6 vs. 160.8±42.7 ms, p=0.003), shorter ejection time (ET) (279.6±20.3 vs. 299.6±34.7 ms, p<0.001) and higher E/A ratio (1.53±0.7 vs. 1.21±0.3, p<0.001). Statistically significantly higher MPI (0.56±0.09 vs. 0.44±0.07, p<0.001), longer IRT (112.3±13.4 vs. 91.8±12.1 ms, p<0.001), longer DT (182.1±30.6 vs. 161.5±43.5 ms, p=0.003), shorter ET 279.6±20.3 vs. 298.8±36.8 ms, p<0.001) and higher E/A ratio (1.53±0.7 vs. 1.22±0.4, p<0.001) were observed during the acute infection period than after clinical recovery. Left ventricular ejection fraction was similar in the controls, during the acute infection period and after clinical recovery.ConclusionsSubclinical diastolic impairment without systolic involvement may be observed in patients with COVID-19. This impairment may be reversible on clinical recovery.     

DEC1基因过表达对人食管癌ECA109细胞增殖及侵袭能力的影响

目的:探讨DEC1基因过表达对人食管癌ECA109细胞增殖和侵袭能力的影响及可能机制。方法:将质粒pc DNA3.1(-)/DEC1(DEC1组)和pc DNA3.1(-)(vector组)利用脂质体分别转染至人食管癌ECA109细胞中,通过real-time PCR检测转染48 h后的细胞内DEC1 mRNA表达,Western blot分别检测转染72 h后细胞内DEC1、基质金属蛋白酶9(MMP9)及细胞周期蛋白cyclin D1的蛋白表达;采用CCK-8实验、平板集落实验及Transwell实验分别检测DEC1过表达对细胞的增殖和侵袭能力的影响。结果:与vector组相比,DEC1组中DEC1的表达明显增高(P0.01);cyclin D1和MMP9的表达明显降低(P0.05);细胞增殖与侵袭能力明显受到抑制(P0.01)。结论:过表达DEC1可明显抑制ECA109细胞的增殖和侵袭能力,DEC1可能通过影响MMP9和cyclin D1参与其中。     

Targeting of rotavirus VP6 to DEC-205 induces protection against the infection in mice

Rotavirus (RV) is the primary etiologic agent of severe gastroenteritis in human infants. Although two attenuated RV-based vaccines have been licensed to be applied worldwide, they are not so effective in low-income countries, and the induced protection mechanisms have not been clearly established. Thus, it is important to develop new generation vaccines that induce long lasting heterotypic immunity. VP6 constitutes the middle layer protein of the RV virion. It is the most conserved protein and it is the target of protective T-cells; therefore, it is a potential candidate antigen for a new generation vaccine against the RV infection. We determined whether targeting the DEC-205 present in dendritic cells (DCs) with RV VP6 could induce protection at the intestinal level. VP6 was cross-linked to a monoclonal antibody (mAb) against murine DEC-205 (αDEC-205:VP6), and BALB/c mice were inoculated subcutaneously (s.c.) twice with the conjugated containing 1.5 μg of VP6 in the presence of polyinosinic–polycytidylic acid (Poly I:C) as adjuvant. As controls and following the same protocol, mice were immunized with ovalbumin (OVA) cross-linked to the mAb anti-DEC-205 (αDEC-205:OVA), VP6 cross-linked to a control isotype mAb (Isotype:VP6), 3 μg of VP6 alone, Poly I:C or PBS. Two weeks after the last inoculation, mice were orally challenged with a murine RV. Mice immunized with α-DEC-205:VP6 and VP6 alone presented similar levels of serum Abs to VP6 previous to the virus challenge. However, after the virus challenge, only α-DEC-205:VP6 induced up to a 45% IgA-independent protection. Memory T-helper (Th) cells from the spleen and the mesenteric lymph node (MLN) showed a Th1-type response upon antigen stimulation in vitro. These results show that when VP6 is administered parenterally targeting DEC-205, it can induce protection at the intestinal level at a very low dose, and this protection may be Th1-type cell dependent.