Implication of a pepper h-type thioredoxin in type I- and II-nonhost resistance to Xanthomonas axonopodis

Thioredoxins (TRXs) are distributed ubiquitously in prokaryotic and eukaryotic organisms. Plants have the most complex forms of TRXs. The functional roles of such TRXs have been studied in abiotic stress but their roles in plant defense responses against biotic stresses have been less well studied. Here, we identified an h-type TRX gene from pepper, CaTRXh1, and characterized its possible effect on Type II nonhost resistance, which entails localized programmed cell death in response to nonhost pathogens. Peptide sequences of CaTRXh1 showed a high degree of similarity with TRXhs from tobacco and Arabidopsis thaliana. Southern blot analyses revealed that CaTRXh1 was present as a single copy in the pepper genome. Intriguingly, leaf infiltration by Xanthomonas axonopodis pv. glycines 8ra, eliciting a visible type II nonhost hypersensitive response (HR), and its type III secretion-system null mutant 8–13, eliciting a type I nonhost non-HR, both induced CaTRXh1 at a level similar to that of pathogenesis-related protein 4, an HR marker gene in pepper. More surprisingly, expression of CaTRXh1 was significantly increased when X. axonopodis pv. vesicatoria race 3 infiltrated the leaf of a pepper cultivar containing a resistance gene, but not with infiltration of a susceptible pepper cultivar. Taken together, our study suggests that the expression of CaTRXh1 has a critical role in HR-mediated active defense responses in pepper. GenBank accession number: EF371503.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Expression of hepatic pyruvate carboxylase mRNA in C57BL/6J Ahb/b and congenic Ahd/d mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The activity and level of hepatic pyruvate carboxylase (PC) has been reported to be altered by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in mice. If alteration in PC level/activity by TCDD influences TCDD toxicity, one would expect to observe an early post-exposure reduction in PC mRNA. To examine the molecular events responsible for the alteration of PC activity in livers of TCDD-treated mice, we designed a synthetic DNA oligonucleotide probe specific for PC mRNA. Northern blot analysis on RNA extracts from hepatic tissue at various times and doses post-TCDD exposure were done. Furthermore, to elucidate the role of the dioxin Ah locus on alterations of PC activity by TCDD, we utilized C57BL/6J (Ah^b/b, Ah high TCDD affinity) mice and a congenic (Ah^d/d, Ah low TCDD affinity) mouse strain. At 8 days post TCDD treatment, a dose-dependent reduction of hepatic PC mRNA levels was observed in Ah^b/b mice. The response, reduction in PC mRNA levels, in the Ah^b/b strain was about 10-fold greater than that of comparably exposed congenic Ah^d/d mice. These results indicate that previously reported reductions in PC activity/level by TCDD treatment of mice is a consequence of a reduction in PC mRNA levels and that the effect requires a competent Ah receptor. © 1996 John Wiley & Sons, Inc.     

Association of ischemic stroke onset time with presenting severity,acute progression,and long-term outcome: A cohort study

BackgroundPreclinical data suggest circadian variation in ischemic stroke progression, with more active cell death and infarct growth in rodent models with inactive phase (daytime) than active phase (nighttime) stroke onset. We aimed to examine the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in human ischemic stroke.Methods and findingsIn a Korean nationwide multicenter observational cohort study from May 2011 to July 2020, we assessed circadian effects on initial stroke severity (National Institutes of Health Stroke Scale [NIHSS] score at admission), END, and favorable functional outcome (3-month modified Rankin Scale [mRS] score 0 to 2 versus 3 to 6). We included 17,461 consecutive patients with witnessed ischemic stroke within 6 hours of onset. Stroke onset time was divided into 2 groups (day-onset [06:00 to 18:00] versus night-onset [18:00 to 06:00]) and into 6 groups by 4-hour intervals. We used mixed-effects ordered or logistic regression models while accounting for clustering by hospitals. Mean age was 66.9 (SD 13.4) years, and 6,900 (39.5%) were women. END occurred in 2,219 (12.7%) patients. After adjusting for covariates including age, sex, previous stroke, prestroke mRS score, admission NIHSS score, hypertension, diabetes, hyperlipidemia, smoking, atrial fibrillation, prestroke antiplatelet use, prestroke statin use, revascularization, season of stroke onset, and time from onset to hospital arrival, night-onset stroke was more prone to END (adjusted incidence 14.4% versus 12.8%, p = 0.006) and had a lower likelihood of favorable outcome (adjusted odds ratio, 0.88 [95% CI, 0.79 to 0.98]; p = 0.03) compared with day-onset stroke. When stroke onset times were grouped by 4-hour intervals, a monotonic gradient in presenting NIHSS score was noted, rising from a nadir in 06:00 to 10:00 to a peak in 02:00 to 06:00. The 18:00 to 22:00 and 22:00 to 02:00 onset stroke patients were more likely to experience END than the 06:00 to 10:00 onset stroke patients. At 3 months, there was a monotonic gradient in the rate of favorable functional outcome, falling from a peak at 06:00 to 10:00 to a nadir at 22:00 to 02:00. Study limitations include the lack of information on sleep disorders and patient work/activity schedules.ConclusionsNight-onset strokes, compared with day-onset strokes, are associated with higher presenting neurologic severity, more frequent END, and worse 3-month functional outcome. These findings suggest that circadian time of onset is an important additional variable for inclusion in epidemiologic natural history studies and in treatment trials of neuroprotective and reperfusion agents for acute ischemic stroke.

Wi-Sun Ryu and colleagues investigate the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in ischemic stroke.     

Advanced Glycation End Products Increase Salivary Gland Hypofunction in d-Galactose-Induced Aging Rats and Its Prevention by Physical Exercise

A declined salivary gland function is commonly observed in elderly people. Advanced glycation end products (AGEs) are believed to contribute to the pathogenesis of aging. Although physical exercise is shown to increase various organ functions in human and experimental models, it is not known whether it has a similar effect in the salivary glands. In the present study, we evaluated the AGEs burden in the salivary gland in the aging process and the protective effect of physical exercise on age-related salivary hypofunction. To accelerate the aging process, rats were peritoneally injected with D-galactose for 6 weeks. Young control rats and d-galactose-induced aging rats in the old group were not exercised. The rats in the physical exercise group ran on a treadmill (12 m/min, 60 min/day, 3 days/week for 6 weeks). The results showed that the salivary flow rate and total protein levels in the saliva of the d-galactose-induced aging rats were reduced compared to those of the young control rats. Circulating AGEs in serum and secreted AGEs in saliva increased with d-galactose-induced aging. AGEs also accumulated in the salivary glands of these aging rats. The salivary gland of aging rats showed increased reactive oxygen species (ROS) generation, loss of acinar cells, and apoptosis compared to young control mice. However, physical exercise suppressed all of these age-related salivary changes. Overall, physical exercise could provide a beneficial option for age-related salivary hypofunction.     

Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype

HuD, an RNA binding protein, plays a role in the regulation of gene expression in certain types of cells, including neuronal cells and pancreatic β-cells, via RNA metabolism. Its aberrant expression is associated with the pathogenesis of several human diseases. To explore HuD-mediated gene regulation, stable cells expressing short hairpin RNA against HuD were established using mouse neuroblastoma Neuro2a (N2a) cells, which displayed enhanced phenotypic characteristics of cellular senescence. Two approaches, RNA immunoprecipitation (RNA IP)-NanoString profiling and cytokine array, were used to subsequently identify a subset of putative HuD targets that act as senescence-associated secretory phenotype (SASP), including C-C motif ligand 2 (CCL2), CCL20, C-X-C motif chemokine ligand 2 (CXCL2), and interleukin-6 (IL-6). Here, we further demonstrated that HuD regulates the expression of CCL2, a SASP candidate upregulated in cells following HuD knockdown, by binding to the 3′-untranslated region (UTR) of Ccl2 mRNA. Downregulation of HuD increased the level of CCL2 in N2a cells and the brain tissues of HuD knockout (KO) mice. Exposure to γ-irradiation induced cellular senescence in N2a cells and HuD knockdown facilitated stress-induced cellular senescence. Our results reveal that HuD acts as a novel regulator of CCL2 expression, and its aberrant expression may contribute to cellular senescence by regulating SASP production.Subject terms: Senescence, RNA quality control     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

A dual signaling cascade that regulates the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor

Ectodomain shedding is an important mechanism to regulate the biological activities of membrane proteins. We focus here on the signaling mechanism of the ectodomain shedding of heparin-binding epidermal growth factor (EGF)-like growth factor (pro HB-EGF). Lysophosphatidic acid (LPA), a ligand for seven-transmembrane G protein-coupled receptors, stimulates the shedding of pro HB-EGF, which constitutes a G protein-coupled receptor-mediated transactivation of the EGF receptor. Experiments using a series of inhibitors and overexpression of mutant forms of signaling molecules revealed that the Ras-Raf-MEK signal is essential for the LPA-induced shedding. In addition, the small GTPase Rac is involved in the LPA-induced shedding, possibly to promote MEK activation. 12-O-Tetradecanoylphorbol-13-acetate is another potent inducer of pro HB-EGF shedding. We also demonstrate that the LPA-induced pathway is distinct from the 12-O-tetradecanoylphorbol-13-acetate-induced pathway and that these pathways constitute a dual signaling cascade that regulates the shedding of pro HB-EGF.