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BACKGROUND: Acute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure. METHODS: Animals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6ß (ATF6ß), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence. RESULTS: The liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells. CONCLUSIONS: The data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF.
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Ferroptosis , Fallo Hepático Agudo , Ratones , Animales , Tioacetamida/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/prevención & control , Transducción de Señal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/efectos adversos , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Necrosis , ARN Interferente Pequeño/efectos adversos , Estrés del Retículo Endoplásmico/genéticaRESUMEN
An 8-week feeding trial was performed to evaluate the effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) supplementation on growth performance and muscle quality of kuruma shrimp (Marsupenaeus japonicas) (initial weight: 2.00 ± 0.01 g) fed a low protein diet. The positive control diet (HP) with 490 g/kg protein and negative control diet (LP) with 440 g/kg protein were formulated. Based on the LP, 0.25, 0.5, 1, 2 and 4 g/kg ß-hydroxy-ß-methylbutyrate calcium were supplemented to design the other five diets named as HMB0.25, HMB0.5, HMB1, HMB2 and HMB4, respectively. Results showed that compared with the shrimp fed LP, the HP, HMB1 and HMB2 groups had significantly higher weight gain and specific growth rate, while significantly lower feed conversion ratio (p < 0.05). Meanwhile, intestinal trypsin activity was significantly elevated in the above three groups than that of the LP group. Higher dietary protein level and HMB inclusion upregulated the expressions of target of rapamycin, ribosomal protein S6 kinase, phosphatidylinositol 3-kinase, and serine/threonine-protein kinase in shrimp muscle, accompanied by the increases in most muscle free amino acids contents. Supplementation of 2 g/kg HMB in a low protein diet improved muscle hardness and water holding capacity of shrimp. Total collagen content in shrimp muscle increased with increasing dietary HMB inclusion. Additionally, dietary inclusion of 2 g/kg HMB significantly elevated myofiber density and sarcomere length, while reduced myofiber diameter. In conclusion, supplementation of 1-2 g/kg HMB in a low protein diet improved the growth performance and muscle quality of kuruma shrimp, which may be ascribed to the increased trypsin activity and activated TOR pathway, as well as elevated muscle collagen content and changed myofiber morphology caused by dietary HMB.
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Acute liver failure (ALF) is life-threatening and often associated with high mortality rates. The aim of the present study was to investigate whether extracellular histone H3 could induce ferroptosis in hepatic macrophages in ALF and explore its potential mechanism. RAW264.7 macrophages and C57BL/6 mice were used in this study. LPS, D-galactosamine (D-Gal), histone H3, histone H3 antibody, NOD2 agonist Muramyl Dipeptide (MDP) and HDAC6-siRNA were administered in this study. The key molecules of ferroptosis, NOD2, HDAC6 and the NF-κb pathway, were detected. In vitro, histone H3 was released into the extracellular environment from cell nucleus after LPS exposure. In addition, histone H3 could induce ferroptosis in RAW264.7 macrophages with increased level of Fe2+ and ROS and decreased levels of GPX4 and GSH. MDP further aggravated ferroptosis in RAW264.7 macrophages stimulated by histone H3, which was accompanied by elevated NOD2, HDAC6, p-P65 and IκBα. HDAC6-siRNA ameliorated ferroptosis in RAW264.7 macrophages induced by histone H3, which was accompanied by decreased levels of HDAC6, p-P65 and IκBα. However, HDAC6-siRNA did not alter NOD2 levels in RAW264.7 macrophages administered histone H3. In vivo, the levels of NOD2, HDAC6 the NF-κb pathway and ferroptosis were increased in ALF mice, which were downregulated by histone H3 antibody and upregulated by histone H3. Extracellular histone H3 could induce ferroptosis in hepatic macrophages in ALF by regulating theNOD2-mediated HDAC6/NF-κb signalling pathway.
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Ferroptosis , Fallo Hepático Agudo , Animales , Ratones , Acetilmuramil-Alanil-Isoglutamina/farmacología , Histonas , Lipopolisacáridos , Fallo Hepático Agudo/inducido químicamente , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Vitamin K (VK), a fatsoluble vitamin, is well known as an anticoagulant in the clinic. It is essential for the posttranslational activation of VKdependent proteins (VKDPs) because hydroquinone VK is a cofactor of glutamine carboxylase. At present, 17 VKDPs are known, which are mainly involved in coagulation and calcification. When Glu residues are carboxylated to Gla residues, these proteins gain a higher calciumbinding ability, which explains why VK has an important role in blood coagulation and biomineralization. However, the current view on the role of VK and several VKDPs in biomineralization remains inconsistent. For instance, conflicting results have been reported regarding the effect of osteocalcin gene knockout on the bone of mice; matrix Gla protein (MGP) promotes osteoblasts mineralization but inhibits vascular smooth muscle cell mineralization. The present review aimed to summarize the existing evidence that several VKDPs, including osteocalcin, MGP, Glarich protein and growth arrest specific 6 are closely related to calcification, including bone health, vascular calcification and lithiasis. The current review discussed these controversies and provided suggestions for future studies on VKDPs, i.e. taking into account dietary habits, geographical environments and genetic backgrounds.
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Calcificación Vascular , Vitamina K , Ratones , Animales , Vitamina K/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Biomineralización , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Calcificación Vascular/genética , Huesos/metabolismoRESUMEN
Sodium dehydroacetate (DHA-S), a potent antifungal and antibacterial agent, is widely used in food, feed and cosmetics. However, recent studies have shown that DHA-S could pose a risk for human and animal health. We had previously reported that DHA-S could cause coagulation disorders in rats and chicken. In the present study, we further confirmed that DHA-S induced blood coagulation via VKORC1 and VKORC1L1 in rats, and elucidated the role played by mTOR/ERK signaling. The in vivo studies demonstrated that PT, APTT, and DHA-S content and relative protein expressions in tissues rebounded after drug withdrawal. In BRL-3A cells, 1.0 mM DHA-S increased the expression levels of mTOR, p-mTOR and p-ERK and decreased the levels of VKORC1, VKORC1L1 and Vitamin K. Rapamycin significantly decreased the expression levels of p-mTOR and p-ERK, while FR180204 (p-ERK Inhibition) lead to a decrease in p-ERK level. Rapamycin and FR180202 attenuated the inhibitory effect of DHA-S on VKORC1, VKORC1L1 and vitamin K levels. In addition, DHA-S increased the expression levels of mTOR, p-mTOR and p-ERK in male and female rat livers and prolonged PT and APTT. In summary, this study indicated that DHA-S induced blood coagulation via the modulation of the mTOR/ERK pathway in rats.
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Coagulación Sanguínea , Sistema de Señalización de MAP Quinasas , Pironas , Humanos , Ratas , Masculino , Femenino , Animales , Vitamina K Epóxido Reductasas/metabolismo , Vitamina K , Serina-Treonina Quinasas TOR/metabolismo , Sirolimus/farmacologíaRESUMEN
The mitochondrial calcium uniporter complex (MCUc), serving as the specific channel for calcium influx into the mitochondrial matrix, is integral to calcium homeostasis and cellular integrity. Given its importance, ongoing research spans various disease models to understand the properties of the MCUc in pathophysiological contexts, but reported a different conclusion. Therefore, this review delves into the profound connection between MCUc-mediated calcium transients and cellular signaling pathways, mitochondrial dynamics, metabolism, and cell death. Additionally, we shed light on the recent advancements concerning the structural intricacies and auxiliary components of the MCUc in both resting and activated states. Furthermore, emphasis is placed on novel extrinsic and intrinsic regulators of the MCUc and their therapeutic implications across a spectrum of diseases. Meanwhile, we employed molecular docking simulations and identified candidate traditional Chinese medicine components with potential binding sites to the MCUc, potentially offering insights for further research on MCUc modulation.
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Canales de Calcio , Calcio , Homeostasis , Mitocondrias , Canales de Calcio/metabolismo , Canales de Calcio/química , Humanos , Calcio/metabolismo , Mitocondrias/metabolismo , Animales , Señalización del CalcioRESUMEN
Object detector based on fully convolutional network achieves excellent performance. However, existing detection algorithms still face challenges such as low detection accuracy in dense scenes and issues with occlusion of dense targets. To address these two challenges, we propose an Global Remote Feature Modulation End-to-End (GRFME2E) detection algorithm. In the feature extraction phase of our algorithm, we introduces the Concentric Attention Feature Pyramid Network (CAFPN). The CAFPN captures direction-aware and position-sensitive information, as well as global remote dependencies of features in deep layers by combining Coordinate Attention and Multilayer Perceptron. These features are used to modulate the front-end shallow features, enhancing inter-layer feature adjustment to obtain comprehensive and distinctive feature representations.In the detector part, we introduce the Two-Stage Detection Head (TS Head). This head employs the First-One-to-Few (F-O2F) module to detect slightly or unobstructed objects. Additionally, it uses masks to suppress already detected instances, and then feeds them to the Second-One-to-Few (S-O2F) module to identify those that are heavily occluded. The results from both detection stages are merged to produce the final output, ensuring the detection of objects whether they are slightly obscured, unobstructed, or heavily occluded. Experimental results on the pig detection dataset demonstrate that our GRFME2E achieves an accuracy of 98.4%. In addition, more extensive experimental results show that on the CrowdHuman dataset, our GRFME2E achieves 91.8% and outperforms other methods.
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This study explores the role of SIRT2 in regulating autophagy and its interaction with AMPK in the context of acute liver failure (ALF). This study investigated the effects of SIRT2 and AMPK on autophagy in ALF mice and TAA-induced AML12 cells. The results revealed that the liver tissue in ALF model group had a lot of inflammatory cell infiltration and hepatocytes necrosis, which were reduced by SIRT2 inhibitor AGK2. In comparison to normal group, the level of SIRT2, P62, MDA, TOS in TAA group were significantly increased, which were decreased in AGK2 treatment. Compared with normal group, the expression of P-PRKAA1, Becilin1 and LC3B-II was decreased in TAA group. However, AGK2 enhanced the expression of P-PRKAA1, Becilin1 and LC3B-II in model group. Overexpression of SIRT2 in AML12 cell resulted in decreased P-PRKAA1, Becilin1 and LC3B-II level, enhanced the level of SIRT2, P62, MDA, TOS. Overexpression of PRKAA1 in AML12 cell resulted in decreased SIRT2, TOS and MDA level and triggered more autophagy. In conclusion, the data suggested the link between AMPK and SIRT2, and reveals the important role of AMPK and SIRT2 in autophagy on acute liver failure.
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Proteínas Quinasas Activadas por AMP , Autofagia , Fallo Hepático Agudo , Sirtuina 2 , Sirtuina 2/metabolismo , Sirtuina 2/genética , Animales , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/inducido químicamente , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Masculino , Hepatocitos/metabolismo , Hepatocitos/patología , Transducción de Señal , Modelos Animales de Enfermedad , Línea Celular , Tioacetamida/toxicidad , Hígado/metabolismo , Hígado/patología , Furanos , QuinolinasRESUMEN
Aim: This study examined the association between self-reported nature exposure and depression among Chinese prisoners, as well as the mediating and moderating effects of meaning in life and callous-unemotional (CU) traits, respectively. Background: Prisoners are more likely to experience depression than any other mental illness. Exposure to nature has been proposed as a highly cost-effective method of treating their depressive symptoms. However, the mechanism underlying the link between nature exposure and depression among prisoners needs further investigation, as the findings may provide new insights into how to address depression in incarcerated populations. Method: Data were collected through a survey conducted in four prisons in southern China from April to May 2022. The participants were 574 prisoners who anonymously completed four questionnaires about nature exposure, meaning in life, depression, and CU traits. Results: The results show that: (1) meaning in life significantly mediates the association between nature exposure and depression, and (2) CU traits moderate the connection between nature exposure and meaning in life. Conclusion: The current study uncovered that prisoners who contact more with the natural environment have a higher meaning in life and lower depression, and individuals with higher CU traits can benefit more from nature exposure.
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The liver is the main organ associated with metabolism. In our previous studies, we identified that the metabolic enzymes malate dehydrogenase 1 (MDH1) and isocitrate dehydrogenase 1 (IDH1) were differentially expressed in ALF. The aim of this study was to explore the changes in the acetylation of MDH1 and IDH1 and the therapeutic effect of histone deacetylase (HDAC) inhibitor in acute liver failure (ALF). Decreased levels of many metabolites were observed in ALF patients. MDH1 and IDH1 were decreased in the livers of ALF patients. The HDAC inhibitor ACY1215 improved the expression of MDH1 and IDH1 after treatment with MDH1-siRNA and IDH1-siRNA. Transfection with mutant plasmids and adeno-associated viruses, identified MDH1 K118 acetylation and IDH1 K93 acetylation as two important sites that regulate metabolism in vitro and in vivo.
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BACKGROUND: Acute liver failure (ALF) is one of the most common life-threatening diseases in adults without previous liver disease. Glycogen synthase kinase 3ß (GSK3ß) is a serine/threonine protein kinase that is widely distributed in the cells. Inhibition of its activity can inhibit cell death and promote autophagy through various pathways, thus providing a protective effect. In this study, we aimed to investigate the effect on ALF after inhibition of GSK3ß and its potential mechanisms. METHODS: D- galactosamine(D-Gal) in combination with lipopolysaccharide(LPS) was used to induce ALF in vitro and in vivo. And then GSK3ß inhibitor TDZD-8 was used to explore the protective effect against ALF. After TDZD-8 treatment TUNEL staining and flow techniques were used to detect the proportion of apoptosis in liver tissues and cells respectively, while western blotting and immunofluorescence assays were performed to detect the expression levels of apoptosis, pyroptosis and necroptosis-related proteins in tissues and cells. In addition, western blotting was performed to explore the specific mechanism of hepatoprotective effect after GSK3ß inhibition to detect the expression levels of TAK1, TRAF6 and HDAC3 after TRAF6 and HDAC3 inhibition alone. The co-localization of TRAF6 and HDAC3 in vitro was detected by immunofluorescence, while the interaction between TRAF6 and HDAC3 was detected by immunoprecipitation assay. RESULTS: Both in vivo and in vitro experiments, GSK3ß inhibitor TDZD-8 can significantly alleviate the progression of ALF. Inhibition of GSK3ß activity could significantly reduce the level of hepatocyte apoptosis, pyroptosis, necroptosis and improve liver dysfunction and tissue damage. Furthermore, we found that hepatocyte TAK1 and TRAF6 levels decreased and HDAC3 levels increased in ALF, whereas inhibition of GSK3ß upregulated TAK1 and TRAF6 levels and decreased HDAC3 expression. CONCLUSION: GSK3ß inhibitor TDZD-8 can prevent the progression of ALF, and its action may involve the TRAF6/HDAC3/TAK1 pathway.
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The COVID-19 pandemic has brought enormous challenges to both employees and organizations all over the world. Previous studies have found high turnover rates among prison officers since the outbreak of COVID-19. This cross-sectional study aimed to investigate the mediating role of job burnout between job demands and turnover intention, as well as the moderating role of the perceived efficacy in overcoming COVID-19 in Chinese prison officers. In total, 1316 prison officers were recruited to complete an online questionnaire between May 2022 and June 2022 (during the COVID-19 pandemic). The bootstrapping approach was used to assess the moderated mediation model in this study. The results showed that prison officers' job demands were positively associated with their turnover intention. Job burnout mediated the relationship between job demands and turnover intention. Perceived efficacy in overcoming COVID-19 moderated the effect of job burnout on turnover intention. Based on these results, suggestions were provided to reduce the high turnover rate of prison officers in public health events like the COVID-19 pandemic.
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Objective: This study examined the prevalence of anxiety and depression-along with the potential risk and protective factors-among Chinese prison officers during the prolonged COVID-19 pandemic. Method: A cross-sectional survey of 1,268 officers from five prisons in western and southern China was administered between June and July 2022. The questionnaires comprised two sections. In the first section, the Generalized Anxiety Disorder-7 (GAD-7) and Patient Health Questionnaire-9 (PHQ-9) were used to evaluate the prevalence of anxiety and depression, respectively, among prison officers. In the second section, the potential influencing factors were examined. Categorical data were compared using χ2 tests and t-tests; binary logistic regression analysis was performed to identify factors associated with anxiety and depression. Results: The prevalence rates of anxiety and depression among the prison officers were 72.6% and 69.8%, respectively. Risk factors for anxiety were older age, being unmarried, work-family conflicts, job demands, and COVID-19 burnout; protective factors were exercise, positive family relationships, and group cohesion. Work-family conflicts, job demands, intolerance of uncertainty regarding COVID-19, and COVID-19 burnout were risk factors for depression, whereas annual income >150,000 RMB, exercise, positive family relationships, group cohesion, and job autonomy were protective factors against depression. Conclusion: The prevalence of anxiety and depression among Chinese prison officers was relatively high during the prolonged COVID-19 pandemic, and more targeted measures should be implemented to improve their mental health. This study offers a reference for improving prison officers' mental health in response to similar public health emergencies in the future.
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Ansiedad , COVID-19 , Personal de Instituciones Correccionales , Depresión , Humanos , Ansiedad/epidemiología , Trastornos de Ansiedad/epidemiología , COVID-19/epidemiología , COVID-19/psicología , Estudios Transversales , Depresión/epidemiología , Pueblos del Este de Asia , Pandemias , Prevalencia , Prisiones/estadística & datos numéricos , Personal de Instituciones Correccionales/psicología , Personal de Instituciones Correccionales/estadística & datos numéricosRESUMEN
PANoptosis is a new cell death proposed by Malireddi et al in 2019, which is characterized by pyroptosis, apoptosis and necroptosis, but cannot be explained by any of them alone. The interaction between pyroptosis, apoptosis and necroptosis is involved in PANoptosis. In this review, from the perspective of PANoptosis, we focus on the relationship between pyroptosis, apoptosis and necroptosis, the key molecules in the process of PANoptosis and the formation of PANoptosome, as well as the role of PANoptosis in diseases. We aim to understand the mechanism of PANoptosis and provide a basis for targeted intervention of PANoptosis-related molecules to treat human diseases.
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The frequent occurrences of cyberbullying on social platforms have sparked a great deal of social conflict, and bystander intervention plays a crucial role in preventing the escalation of cyberbullying. This research examines the impact of victim self-disclosure on bystander intervention in cyberbullying through two experimental studies. The studies collected data from March to July of 2022, utilizing a convenience sampling approach to recruit university students as experiment participants. Study 1 recruited 247 valid participants, while Study 2 recruited 522 eligible participants. The results of Study 1 indicate that the perceptible dimensions (frequency, privacy, and valence) of victim self-disclosure impact bystander intervention. Specifically, in a low privacy context, positive self-disclosure increases bystander intervention, while negative self-disclosure does the opposite. The results of Study 2 suggest that the valence of self-disclosure affects bystander intervention through the mediation of victim blaming, with interpersonal distance moderating the impact of victim self-disclosure valence on the extent of victim blaming. This moderated mediation model clarifies the psychological process by which the valence of victim self-disclosure affects bystander intervention. The findings of this study contribute to the understanding of the social psychological process behind bystander intervention, providing a scientific basis and pathway for reducing cyberbullying and fostering a harmonious online environment.
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Over the past two decades, non-alcoholic fatty liver disease (NAFLD) has become a leading burden of hepatocellular carcinoma and liver transplantation. Although the exact pathogenesis of NAFLD has not been fully elucidated, recent hypotheses placed more emphasis on the crucial role of the gut microbiome and its derivatives. Reportedly, microbial metabolites such as short-chain fatty acids, amino acid metabolites (indole and its derivatives), bile acids (BAs), trimethylamine N-oxide (TMAO), and endogenous ethanol exhibit sophisticated bioactive properties. These molecules regulate host lipid, glucose, and BAs metabolic homeostasis via modulating nutrient absorption, energy expenditure, inflammation, and the neuroendocrine axis. Consequently, a broad range of research has studied the therapeutic effects of microbiota-derived metabolites. In this review, we explore the interaction of microbial products and NAFLD. We also discuss the regulatory role of existing NAFLD therapies on metabolite levels and investigate the potential of targeting those metabolites to relieve NAFLD.
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RNA silencing is a conserved mechanism in eukaryotic organisms to regulate gene expression. Argonaute (AGO), Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) proteins are critical components of RNA silencing, but how these gene families' functions in sugarcane were largely unknown. Most stress-resistance genes in modern sugarcane cultivars (Saccharum spp.) were originated from wild species of Saccharum, for example S. spontaneum. Here, we used genome-wide analysis and a phylogenetic approach to identify four DCL, 21 AGO and 11 RDR genes in the S. spontaneum genome (termed SsDCL, SsAGO and SsRDR, respectively). Several genes, particularly some of the SsAGOs, appeared to have undergone tandem or segmental duplications events. RNA-sequencing data revealed that four SsAGO genes (SsAGO18c, SsAGO18b, SsAGO10e and SsAGO6b) and three SsRDR genes (SsRDR2b, SsRDR2d and SsRDR3) tended to have preferential expression in stem tissue, while SsRDR5 was preferentially expressed in leaves. qRT-PCR analysis showed that SsAGO10c, SsDCL2 and SsRDR6b expressions were strongly upregulated, whereas that of SsAGO18b, SsRDR1a, SsRDR2b/2d and SsRDR5 was significantly depressed in S. spontaneum plants exposed to PEG-induced dehydration stress or infected with Xanthomonas albilineans, causal agent of leaf scald disease of sugarcane, suggesting that these genes play important roles in responses of S. spontaneum to biotic and abiotic stresses.
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Proteínas Argonautas/genética , Estudio de Asociación del Genoma Completo , ARN Polimerasa Dependiente del ARN/genética , Ribonucleasa III/genética , Saccharum/genética , Cromosomas de las Plantas/genética , Simulación por Computador , Regiones Promotoras Genéticas/genética , Mapeo de Interacción de Proteínas , Saccharum/enzimología , Saccharum/metabolismoRESUMEN
In the studies of expressing various foreign peptides using a TMV-based vector, a portion of morphologically altered progeny viral particles from some recombinant TMV constructs were detected by transmission electron microscopy in the first systematically infected upper leaves, but not in the fully expanded inoculated leaves, from infected tobacco plants. Furthermore, in vitro stability of such recombinant TMV constructs were lower than those of the wild type and other recombinant TMV constructs able to form regular rod-shape virions, hence causing the lower yields of recombinant viral particles purified from the infected tobacco plants. Our studies revealed that the presence of a cysteine residue in the foreign peptides, regardless of its position and the peptide sequence, was directly related to changes in the morphology and stability of these TMV recombinants.
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Proteínas de la Cápside/genética , Cisteína/química , Nicotiana/virología , Virus del Mosaico del Tabaco/genética , Virión/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Péptidos/química , Péptidos/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Virus del Mosaico del Tabaco/metabolismo , Virus del Mosaico del Tabaco/ultraestructura , Virión/química , Virión/genéticaRESUMEN
Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed.