Perspectives of Licorice Production in Harsh Environments of the Aral Sea Regions.

Along with pharmacological applications due to bioactive elements such as flavonoids and glycyrrhizin, licorice has positive influences on the rehabilitation, rejuvenation, and management of salt-affected degraded lands in arid regions. These features made this plant widely appreciated worldwide when climate change is showing detrimental impacts for crop production and food security. However, a growing demand followed by irrational harvesting of wild licorice plants has led to substantial dwindling of its natural habitat. There is an increasing need to protect the plant biodiversity since sustainability can be a problem with wild harvesting. Therefore, it is important to investigate cultivation technologies of licorice under harsh environments, while this plant can adapt to a wide range of climates. Thus, in this review, we studied, analyzed and summarized the literature on licorice cultivation methods counteracting the most common environmental stresses in the Aral Sea region. Particularly, the current knowledge was rationalized regarding on cultivation technologies for alleviating salt stress thereby improving crop production. We also highlighted that future research directions on licorice breeding and genomics that might facilitate to produce more resilient and sustainable licorice genotypes to renovate agricultural productivity under disastrous ecology and climate change of the arid regions. Whereas this area possesses all prerequisite conditions needed for successful cultivation of the alternative cash crop.

Meta-analysis of the efficacy of glycyrrhizin for postoperative liver preservation in patients with liver cancer.

Objective: This meta-analysis comprehensively summarizes the current clinical research on compound glycyrrhizin (CG) treatment for liver cancer and protecting liver function to guide clinical treatment. Methods: Eighteen English-language articles were retrieved from PubMed, SinoMed, Cochrane, Embase, Web of Science, and three Chinese databases: The Wan Fang database, China National Knowledge Infrastructure (CNKI), and the VIP database. Results: CG treatment improved the patient's alanine aminotransferase (ALT) level (in the metastatic liver cancer group: mean deviation (MD) = -13.78, 95% confidence interval (CI) = [-17.29, 10.27]; in the primary liver cancer group: MD = -32.15, 95% CI = [-35.48, 28.81]); aspartate aminotransferase (AST) level (in the primary liver cancer group: MD = -21.63, 95% CI = [-24.29, 18.96]; in the metastatic liver cancer group: MD = -15.64, 95% CI = [-19.08, -12.20]); serum total bilirubin (TBIL) level (MD = -1.61, 95% CI = [-2.71, -0.51]); and serum albumin (ALB) level (MD = 2.80, 95% CI = [1.85, 3.74]). CG treatment was efficient than the control (relative risk [RR] = 1.66, 95% CI = [1.35, 2.04]). Although adverse reactions, including fever, were higher than in the control group (RR = 1.13, 95% CI = [0.89, 1.43]), they were controllable. Conclusion: CG affects liver preservation in treating liver cancer, which can reduce ALT, AST, and TBIL levels in patients; increase the ALB level; and protect liver cells. The CG-treated group showed improvement compared with the control group; although adverse reactions occurred in the treated group, the duration was shortened.

Combined Metabolome and Transcriptome Analyses of Young, Mature, and Old Rhizome Tissues of Zingiber officinale Roscoe.

Ginger (Zingiber officinale Roscoe) is known for its unique pungent taste and useability in traditional Chinese medicine. The main compounds in ginger rhizome can be classified as gingerols, diarylheptanoids, and volatile oils. The composition and concentrations of the bioactive compounds in ginger rhizome might vary according to the age of the rhizome. In this regard, the knowledge on the transcriptomic signatures and accumulation of metabolites in young (Y), mature (M), and old (O) ginger rhizomes is scarce. This study used HiSeq Illumina Sequencing and UPLC-MS/MS analyses to delineate how the expression of key genes changes in Y, M, and O ginger rhizome tissues and how it affects the accumulation of metabolites in key pathways. The transcriptome sequencing identified 238,157 genes of which 13,976, 11,243, and 24,498 were differentially expressed (DEGs) in Y vs. M, M vs. O, and Y vs. O, respectively. These DEGs were significantly enriched in stilbenoid, diarylheptanoid, and gingerol biosynthesis, phenylpropanoid biosynthesis, plant-hormone signal transduction, starch and sucrose metabolism, linoleic acid metabolism, and α-linoleic acid metabolism pathways. The metabolome profiling identified 661 metabolites of which 311, 386, and 296 metabolites were differentially accumulated in Y vs. M, Y vs. O, and M vs. O, respectively. These metabolites were also enriched in the pathways mentioned above. The DEGs and DAMs enrichment showed that the gingerol content is higher in Y rhizome, whereas the Y, M, and O tissues differ in linoleic and α-linoleic acid accumulation. Similarly, the starch and sucrose metabolism pathway is variably regulated in Y, M, and O rhizome tissues. Our results showed that ginger rhizome growth slows down (Y > M > O) probably due to changes in phytohormone signaling. Young ginger rhizome is the most transcriptionally and metabolically active tissue as compared to M and O. The transitioning from Y to M and O affects the gingerol, sugars, linoleic acid, and α-linoleic acid concentrations and related gene expressions.

Terpenoid indole alkaloid biosynthesis in Catharanthus roseus: effects and prospects of environmental factors in metabolic engineering.

Plants synthesize a vast array of specialized metabolites that primarily contribute to their defense and survival under adverse conditions. Many of the specialized metabolites have therapeutic values as drugs. Biosynthesis of specialized metabolites is affected by environmental factors including light, temperature, drought, salinity, and nutrients, as well as pathogens and insects. These environmental factors trigger a myriad of changes in gene expression at the transcriptional and posttranscriptional levels. The dynamic changes in gene expression are mediated by several regulatory proteins that perceive and transduce the signals, leading to up- or down-regulation of the metabolic pathways. Exploring the environmental effects and related signal cascades is a strategy in metabolic engineering to produce valuable specialized metabolites. However, mechanistic studies on environmental factors affecting specialized metabolism are limited. The medicinal plant Catharanthus roseus (Madagascar periwinkle) is an important source of bioactive terpenoid indole alkaloids (TIAs), including the anticancer therapeutics vinblastine and vincristine. The emerging picture shows that various environmental factors significantly alter TIA accumulation by affecting the expression of regulatory and enzyme-encoding genes in the pathway. Compared to our understanding of the TIA pathway in response to the phytohormone jasmonate, the impacts of environmental factors on TIA biosynthesis are insufficiently studied and discussed. This review thus focuses on these aspects and discusses possible strategies for metabolic engineering of TIA biosynthesis. PURPOSE OF WORK: Catharanthus roseus is a rich source of bioactive terpenoid indole alkaloids (TIAs). The objective of this work is to present a comprehensive account of the influence of various biotic and abiotic factors on TIA biosynthesis and to discuss possible strategies to enhance TIA production through metabolic engineering.

Assessment of the Cytoprotective Effects of High-Dose Valproic Acid Compared to a Clinically Used Lower Dose.

OBJECTIVE: Valproic acid (VPA) treatment improves survival in animal models of injuries on doses higher than those allowed by Food and Drug Administration (FDA). We investigated the proteomic alterations induced by a single high-dose (140mg/kg) of VPA (VPA140) compared to the FDA-approved dose of 30mg/kg (VPA30) in healthy humans. We also describe the proteomic and transcriptomic changes induced by VPA140 in an injured patient. We hypothesized that VPA140 would induce cytoprotective changes in the study participants. METHODS: Serum samples were obtained from healthy subjects randomized to two groups; VPA140 and VPA30 at 3 timepoints: 0h(baseline), 2h, and 24h following infusion(n = 3/group). Samples were also obtained from an injured patient that received VPA140 at 0h, 6h and 24h following infusion. Proteomic analyses were performed using liquid chromatography-mass spectrometry (LC-MS/MS), and transcriptomic analysis was performed using RNA-sequencing. Differentially expressed (DE) proteins and genes were identified for functional annotation and pathway analysis using iPathwayGuide and gene set enrichment analysis (GSEA), respectively. RESULTS: For healthy individuals, a dose comparison was performed between VPA140 and VPA30 groups at 2 and 24 h. Functional annotation showed that top biological processes in VPA140 versus VPA30 analysis at 2 h included regulation of fatty acid (P = 0.002) and ATP biosynthesis (P = 0.007), response to hypoxia (P = 0.017), cell polarity regulation (P = 0.031), and sequestration of calcium ions (P = 0.031). Top processes at 24 h in VPA140 versus VPA30 analysis included amino acid metabolism (P = 0.023), collagen catabolism (P = 0.023), and regulation of protein breakdown (P = 0.023). In the injured patient, annotation of the DE proteins in the serum showed that top biological processes at 2 h included neutrophil chemotaxis (P = 0.002), regulation of cellular response to heat (P = 0.008), regulation of oxidative stress (P = 0.008) and regulation of apoptotic signaling pathway (P = 0.008). Top biological processes in the injured patient at 24 h included autophagy (P = 0.01), glycolysis (P = 0.01), regulation of apoptosis (P = 0.01) and neuron apoptotic processes (P = 0.02). CONCLUSIONS: VPA140 induces cytoprotective changes in human proteome not observed in VPA30. These changes may be responsible for its protective effects in response to injuries.

Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza.

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments.

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), ß-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

[Molecular genetics of medicinal plants, past achievement, and new era].

Unraveling the genetic basis of medicinal plant metabolism and developmental traits is a long-standing goal for pharmacologists and plant biologists. This paper discusses the definition of molecular genetics of medicinal plants, which is an integrative discipline with medicinal plants as the research object. This discipline focuses on the heredity and variation of medicinal plants, and elucidates the relationship between the key traits of medicinal plants(active compounds, yield, resistance, etc.) and genotype, studies the structure and function, heredity and variation of medicinal plant genes mainly at molecular level, so as to reveal the molecular mechanisms of transmission, expression and regulation of genetic information of medicinal plants. Specifically, we emphasize on three major aspects of this discipline.(1)Individual and population genetics of medicinal plants, this part mainly highlights the genetic mechanism of the domestication, the individual genomics at the species level, and the formation of genetic diversity of medicinal plants.(2)Elucidation of biosynthetic pathways of active compounds and their evolutionary significance. This part summarizes the biosynthesis, diversity and molecular evolution of active compounds in medicinal plants.(3) Molecular mechanisms that shaping the key agronomic traits by internal and external factors. This part focuses on the accumulation and distribution of active compounds within plants and the regulation of metabolic network by environmental factors. Finally, we prospect the future direction of molecular genetics of medicinal plants based on the rapid development of multi-omics technology, as well as the application of molecular genetics in the future strategies to achieve conservation and breeding of medicinal plants and efficient biosynthesis of active compounds.

Mitochondrial Pyruvate Carriers Prevent Cadmium Toxicity by Sustaining the TCA Cycle and Glutathione Synthesis.

Cadmium (Cd) is a major heavy metal pollutant, and Cd toxicity is a serious cause of abiotic stress in the environment. Plants protect themselves against Cd stress through a variety of pathways. In a recent study, we found that mitochondrial pyruvate carriers (MPCs) are involved in Cd tolerance in Arabidopsis (Arabidopsis thaliana). Following the identification of MPCs in yeast (Saccharomyces cerevisiae) in 2012, most studies have focused on the function of MPCs in animals, as a possible approach to reduce the risk of cancer developing. The results of this study show that AtMPC protein complexes are required for Cd tolerance and prevention of Cd accumulation in Arabidopsis. AtMPC complexes are composed of two elements, AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). When the formation of AtMPCs was interrupted by the loss of AtMPC1, glutamate could supplement the synthesis of acetyl-coenzyme A and sustain the TCA cycle. With the up-regulation of glutathione synthesis following exposure to Cd stress, the supplementary pathway could not efficiently drive the tricarboxylic acid cycle without AtMPC. The ATP content decreased concomitantly with the deletion of tricarboxylic acid activity, which led to Cd accumulation in Arabidopsis. More importantly, ScMPCs were also required for Cd tolerance in yeast. Our results suggest that the mechanism of Cd tolerance may be similar in other species.

Addition of low-dose valproic acid to saline resuscitation provides neuroprotection and improves long-term outcomes in a large animal model of combined traumatic brain injury and hemorrhagic shock.

BACKGROUND: Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) is highly lethal. In a nonsurvival model of TBI + HS, addition of high-dose valproic acid (VPA) (300 mg/kg) to hetastarch reduced brain lesion size and associated swelling 6 hours after injury; whether this would have translated into better neurologic outcomes remains unknown. It is also unclear whether lower doses of VPA would be neuroprotective. We hypothesized that addition of low-dose VPA to normal saline (NS) resuscitation would result in improved long-term neurologic recovery and decreased brain lesion size. METHODS: TBI was created in anesthetized swine (40-43 kg) by controlled cortical impact, and volume-controlled hemorrhage (40% volume) was induced concurrently. After 2 hours of shock, animals were randomized (n = 5 per group) to NS (3× shed blood) or NS + VPA (150 mg/kg). Six hours after resuscitation, packed red blood cells were transfused, and animals were recovered. Peripheral blood mononuclear cells were analyzed for acetylated histone-H3 at lysine-9. A Neurological Severity Score (NSS) was assessed daily for 30 days. Brain magnetic resonance imaging was performed on Days 3 and 10. Cognitive performance was assessed by training animals to retrieve food from color-coded boxes. RESULTS: There was a significant increase in histone acetylation in the NS + VPA-treated animals compared with NS treatment. The NS + VPA group demonstrated significantly decreased neurologic impairment and faster speed of recovery as well as smaller brain lesion size compared with the NS group. Although the final cognitive function scores were similar between the groups, the VPA-treated animals reached the goal significantly faster than the NS controls. CONCLUSION: In this long-term survival model of TBI + HS, addition of low-dose VPA to saline resuscitation resulted in attenuated neurologic impairment, faster neurologic recovery, smaller brain lesion size, and a quicker normalization of cognitive functions.

Protective effect of suberoylanilide hydroxamic acid against lipopolysaccharide-induced liver damage in rodents.

BACKGROUND: Lipopolysaccharide (LPS) has a deleterious effect on several organs, including the liver, and eventually leads to endotoxic shock and death. LPS-induced hepatotoxicity is characterized by disturbed intracellular redox balance and excessive reactive oxygen species (ROS) accumulation, leading to liver injury. We have shown that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improves survival in a murine model of LPS-induced shock, but the protective effect of SAHA against liver damage remains unknown. The goal of this study was to investigate the mechanism underlying SAHA action in murine livers. METHOD: Male C57BL/6J mice (6-8 wk), weighing 20-25 g, were randomly divided into three groups: (A) a sham group was given isotonic sodium chloride solution (10 µL/g body weight, intraperitoneal, i.p.) with dimethyl sulfoxide (DMSO; 1 µL/g body weight, i.p.); (B) an LPS group was challenged with LPS (20 mg/kg, i.p.) dissolved in isotonic sodium chloride solution with DMSO; (C) and an LPS plus SAHA group was treated with SAHA (50 mg/kg, i.p.) dissolved in DMSO immediately after injection of LPS (20 mg/kg, i.p.). Mice were anesthetized, and their livers were harvested 6 or 24 h after injection to analyze whether SAHA affected production of ROS and activation of apoptotic proteins in the liver cells of challenged mice. RESULTS: SAHA counteracted LPS-induced production of ROS (thiobarbituric acid reactive substances and nitrite) and reversed an LPS-induced decrease in antioxidant enzyme, glutathione. SAHA also attenuated LPS-induced hepatic apoptosis. Moreover, SAHA inhibited activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1, and the mitogen-activated protein kinases, p38 and Jun N-terminal kinase. CONCLUSIONS: Our data indicate, for the first time, that SAHA is capable of alleviating LPS-induced hepatotoxicity and suggest that a blockade of the upstream events required for apoptosis signal-regulating kinase-1 action may serve as a new therapeutic option in the treatment of LPS-induced inflammatory conditions.

Pharmacologic modulation of cerebral metabolic derangement and excitotoxicity in a porcine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Cerebral metabolic derangement and excitotoxicity play critical roles in the evolution of traumatic brain injury (TBI). We have shown previously that treatment with large doses of valproic acid (VPA) decreases the size of brain lesion. The goal of this experiment was to determine whether this effect was owing to metabolic modulation. METHODS: Yorkshire swine (n = 9) underwent a protocol of computer-controlled TBI and 40% hemorrhage and were resuscitated randomly with either fresh frozen plasma equal to the volume of shed blood (FFP; n = 4) or VPA (300 mg/kg) and FFP (FFP+VPA; n = 5). Hemodynamics, brain oxygenation, and blood glucose were monitored continuously for 6 hours after resuscitation. Cerebral microdialysis was used to measure glucose, lactate, pyruvate, glutamate, and glycerol levels at baseline, 1 and 2 hours post-shock, post-resuscitation (PR), and at 2, 4, and 6 hours PR. Brain samples from the injured side were then separated into mitochondrial and cytosolic fractions, and activity of pyruvate dehydrogenase complex (PDH) was measured using a dipstick assay kit. RESULTS: At baseline, there was no difference in brain lactate, pyruvate, glycerol, and glutamate concentrations between the groups. At all time points, there were no differences between the groups in brain oxygenation, cerebral perfusion pressure, or blood and brain glucose concentrations. After VPA infusion (PR time point), however, there was sustained decrease in lactate (0.91 ± 0.47 vs 2.54 ± 0.59 mmol/L; P < .01) and pyruvate (12.80 ± 4.89 vs 46.25 ± 9.22; P < .001) concentrations compared with the FFP alone group, implying superior glucose utilization for ATP production. There was also a decrease in concentrations of glutamate (6.64 ± 3.68 vs 42.25 ± 27.07 mmol/L; P = .02) and glycerol (19.20 ± 6.76 vs 69.75 ± 30.07 mmol/L; P = .01), in the FFP+VPA group, signifying lesser degree of excitotoxicity and brain damage, respectively. Brain PDH activity was greater in the mitochondrial fractions (5,984 ± 504 adjusted volume intensity [INT] × mm(2) vs 4,332 ± 1,055 INT × mm(2); P = .04) and lower in cytosolic fractions in the FFP+VPA group (1,597 ± 1,395 vs 4,026 ± 1,067 INT × mm(2); P = .03), indicating better mitochondrial membrane function and enhanced mitochondrial PDH retention. CONCLUSION: VPA treatment attenuates perturbation of post-traumatic cerebral metabolism by mitigating mitochondrial dysfunction, and decreases glutamate-mediated excitotoxic damage. These properties could explain its effectiveness in decreasing lesion size and post-traumatic cerebral edema.

Synergistic effects of hypertonic saline and valproic acid in a lethal rat two-hit model.

BACKGROUND: Hemorrhagic shock (HS) followed by an infection ("second hit") can lead to severe systemic inflammatory response and multiple-organ failure. Studies have shown that resuscitation with hypertonic saline (HTS) can blunt the inflammatory response. We demonstrated that large doses of valproic acid (VPA, 300 mg/kg), a histone deacetylase inhibitor, improves survival in a rodent two-hit model (HS followed by cecal ligation and puncture [CLP]). In the present study, we examined whether combination of HTS with VPA would allow us to achieve survival advantage at a lower dose of VPA (200 mg/kg). METHODS: Male Sprague-Dawley rats were subjected to HS (50% blood loss) and randomized into five groups (n = 7-8 per group) as follows: (1) isotonic sodium chloride solution (ISCS), (2) 7.5% saline, (3) VPA, (4) ISCS + VPA, and (5) HTS + VPA. After 24 hours, they underwent CLP, followed by the same doses of ISCS, HTS, and/or VPA and were monitored for 10 days. In a parallel experiment, blood, peritoneal irrigation fluid and lung homogenate were subjected to enzyme-linked immunosorbent assay 3 hours and 24 hours after CLP to measure myeloperoxidase activity and proinflammatory cytokines tumor necrosis factor α and interleukin 1ß levels. Western blotting was performed to investigate the expression of pentraxin 3 protein in the lung homogenate at 24 hours after CLP. Hematoxylin and eosin staining of lungs at the 24 hours were performed to quantify the degree of acute lung injury. RESULTS: HTS + VPA treatment significantly improved survival (87.5%), compared with the other groups (14.3%; p < 0.05), while attenuating peritoneal myeloperoxidase levels and proinflammatory cytokine tumor necrosis factor α and interleukin 1ß levels in the serum, peritoneal cavity, and lung. The degree of acute lung injury and expression of pentraxin 3 in the lung were significantly reduced in the HTS + VPA group. CONCLUSION: This is the first study to show that VPA and HTS can work synergistically to attenuate inflammation and improve survival in a lethal two-hit model.

Prevention of hypoxia-induced neuronal apoptosis through histone deacetylase inhibition.

BACKGROUND: We have recently discovered that administration of valproic acid (VPA), a histone deacetylase inhibitor, enhances nuclear histone acetylation and improves survival after lethal hemorrhage in rats. In the present study, neurons were subjected to severe hypoxic condition in vitro to test whether VPA would prevent hypoxia-induced apoptosis, and to explore the possible mechanisms. METHODS: Primary hippocampal and cortical cultures dissociated from E18 rat embryos were plated in quadruplicate at a density of 2 x 10/well in neurobasal medium supplemented with B-27 on glass cover-slips coated with poly-l-lysine. On the 10th day after plating, cells were incubated in a hypoxia chamber (0.5% O2, 10% CO2, 89.5% N2) at 37 degrees C for 6 hour and 16 hour in the presence or absence of VPA (1 mmol/L). The cells were then fixed, stained with antiactivated caspase-3 and antiacetyl histone H3 lysine 9 (Ac H3 K9) antibodies and visualized under confocal microscope. The caspase-3 positive cells were counted as apoptotic. Ratio of the apoptotic to total cells stained with 4',6-diamidino-2-phenylindole was determined. Numerical data were subjected to t test analysis. p < 0.05 was considered statistically significant. Western blot was performed to determine the level of acetylation of nuclear factor-kappa B (NF-kappaB) and phospho-JNK (c-Jun N-terminal kinase) in cells treated with or without VPA. Luciferase report assay was employed to analyze the activation of NF-kappaB after the cells were transfected with NF-kBLuc with or without VPA treatment. RESULTS: Exposure of neurons to VPA prevented apoptotic cell death under hypoxic conditions (20% apoptosis). In contrast, about 95% cells underwent apoptosis at the same level of hypoxia. VPA treatment induced acetylation of histone H3 K9 and NF-kappaB lysine 310. NF-kappaB was activated at the same time as the protein acetylation. Moreover, JNK phosphorylation was inhibited after the cells were treated with VPA under hypoxia condition. CONCLUSION: VPA enhances acetylation of histone 3 at lysine 9 and NF-kappaB at 310, induces NF-kappaB activation, reduces JNK activation, and protects the neurons from hypoxia-induced apoptosis in vitro.

ZNF325, a novel human zinc finger protein with a RBaK-like RB-binding domain, inhibits AP-1- and SRE-mediated transcriptional activity.

Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. The zinc-finger-containing transcription factors have been previously revealed to be involved in the regulation of the MAPK signaling pathways. Here, we have identified a novel human zinc-finger transcriptional repressor, ZNF325, that contains a RBaK-like RB-binding domain and 15 tandem repeated C2H2 type zinc fingers. Northern blot analysis indicates that a 2.7 kb transcript specific for ZNF325 is widely expressed in all tissues examined at adult stage and in most of the embryonic tissues. Overexpression of ZNF325 in COS-7 cells inhibits the transcriptional activities of AP-1 and SRE. The deletion and RNAi analysis indicate that the C2H2 zinc finger motifs represent the basal transcriptional repressive activity. These results indicate that the ZNF325 protein may act as a novel transcription repressor in MAPK signaling pathway to mediate cellular functions.

ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1.

The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C2H2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions.

A novel KRAB zinc-finger protein, ZNF480, expresses in human heart and activates transcriptional activities of AP-1 and SRE.

The zinc-finger motif found in many transcription factors is thought to be important for human heart development and diseases. In this study, we have identified and characterized a novel zinc-finger gene named ZNF480 using degenerate primers from an early human embryo heart cDNA library. ZNF480 contains a KRAB-A box and 12 C2H2 zinc fingers. The cDNA sequence contains an open reading frame of 1551 bp, encoding a putative protein of 516 amino acid residues with a predicted molecular mass of 57 kDa. Northern blot analysis indicates that a 4.7kb transcript specific for ZNF480 is expressed only in embryonic heart. In the adult tissues, the expression of ZNF480 is restricted largely to heart, skeletal muscle, pancreas, and placenta. Overexpression of ZNF480 in cells activates the transcriptional activities of AP-1 and SRE. Therefore, our data suggest that ZNF480 may act as a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.

A novel human SCAN/(Cys)2(His)2 zinc-finger transcription factor ZNF323 in early human embryonic development.

The C(2)H(2) zinc-finger motif found in many transcription factors is thought to be important for nucleic acid binding and/or dimerization. Here, we have identified and characterized a novel zinc-finger gene named ZNF323 using degenerate primers from an early human embryo heart cDNA library. The predicted protein contains six different C(2)H(2) type zinc fingers and a SCAN box. ZNF323 maps to chromosome 6p22.1-22.3. The expression levels were different during different development stages of human embryo between 15 and 23 weeks. Northern blot analysis shows that a 3.2-kb transcript specific for ZNF323 was expressed at high levels in the lung, liver, and kidney, while weakly expressed in intestine, brain, muscle, cholecyst, heart, and pancreas. In adult tissues, ZNF323 is expressed at high levels in liver and kidney, weakly in lung, pancreas, brain, placenta, muscle, and heart. Taken together, these results indicate that ZNF323 is a member of the zinc-finger transcription factor family and may be involved in the development of multiple embryonic organs.

Identification and characterization of two novel zinc finger genes, ZNF359 and ZFP28, in human development.

Transcription factors play an essential role in controlling gene expression during cardiac and vascular pathogeneses. Identification of regulatory genes in the cardiovascular system is a necessary step toward an understanding of the pathogenesis of congenital heart disease and acquired cardiovascular diseases. The Cys2/His2 type zinc finger genes are the single largest class of transcription factors in the human genome and many numbers of these krüpple-like zinc finger genes have been found to be involved in cardiac development or cardiovascular diseases. In this study, we have identified two novel human krüpple-like zinc finger genes named ZNF359 and ZFP28 from the human heart cDNA library. The complete human ZNF359 cDNA sequence is 3270bp and contains a 1932-bp open reading frame (ORF) that encodes a 643 amino acid protein with an N-terminal KRAB domain and 16 C-terminus zinc finger C2H2 motifs. The ZFP28 cDNA sequence is 4104bp and contains a 2076-bp ORF that encodes an 868 amino acid protein with an N-terminal signal peptide, two KRAB domains, and 14 C-terminal C2H2 zinc finger motifs. Northern blot analyses showed a strong expression of ZNF359 and ZFP28 in various tissues of adult human. A further analysis using human embryonic tissues (18-23 weeks) showed a development-specific expression pattern in heart, skeletal muscle, liver, lung, kidney, and brain, suggesting a role for these genes in embryonic development.