Minor immunosuppressive spiroorthoester group-containing pregnane glycosides from the root barks of Periploca sepium.

LC-MS guided chemical investigation of the periploside-rich extract of the root barks of Periploca sepium afforded six new minor pregnane glycosides, named periplosides A1-A6 (1-6). Their structures were characterized on the basis of extensive spectroscopic analysis. Compounds 1-6 were evaluated for their inhibitory activities against the proliferation of T and B lymphocytes in vitro, among them, compound 5 exhibited significant inhibitory activities and the most favorite selective index (SI) values against the proliferation of T lymphocyte (IC50 = 0.30 µM, SI = 176) and B lymphocyte (IC50 = 0.55 µM, SI = 97).

[Mechanism of lysosome-mediated eradication activity of macrophages on specific anti-Helicobacter pylori of patchouli alcohol].

The aim of this paper is to investigate the effect of patchouli alcohol in enhancing Helicobater pylori's action in eradicating macrophages and its mechanism. H. pylori was co-cultured with macrophages at a ratio of MOI=100 in different concentrations of patchouli alcohol. The effect of patchouli alcohol in eradicating macrophages was detected by agar dilution method. The effect of patchouli alcohol on NO and myeloperoxidase (MPO) levels in macrophages were measured by H. pylori by biochemical methods. Patchouli alcohol effect on H. pylori-induced pro-inflammatory gene expression and protein secretion in macrophages were detected by RT-qPCR and ELISA method. The eradication of H. pylori has significantly enhanced, and the destabilization of lysosomes has been reversed. Meanwhile, patchouli alcohol has an effect in inhibiting pro-inflammation and oxidation. The mechanism of patchouli alcohol in eradicating H. pylori and resisting oxidative stress may be associated to the blocking of bacteria escape lysosome combination procedures.

Activated Circulating Myeloid-Derived Suppressor Cells in Patients with Dilated Cardiomyopathy.

BACKGROUND/AIMS: Myeloid-derived suppressor cells (MDSCs) are increased in inflammatory and autoimmune disorders. This study aims to evaluate the significance of MDSCs in dilated cardiomyopathy (DCM) patients. METHODS: In total, 42 newly hospitalized DCM patients and 39 healthy controls were enrolled in the study. The frequencies of circulating CD14+HLA-DR-/low MDSCs were determined by flow cytometry. Then, the functional properties of MDSCs in suppressing T cell proliferation and interferon-gamma (IFN-x03B3;) production were measured in a co-culture model. Then, mRNA expression levels of various important molecules in peripheral blood mononuclear cells were measured by real time polymerase chain reaction. Furthermore, correlation analyses between MDSC frequencies and cardiac function parameters were also performed. RESULTS: The frequencies of circulating CD14+HLA-DR-/low MDSCs were significantly elevated in DCM patients compared with healthy controls. It showed that MDSCs from DCM patients more effectively suppressed T cell proliferation and IFN-x03B3; production compared with those from healthy controls, which was partially mediated by arginase-1 (Arg-1). In addition, the correlation analysis suggested that MDSC frequencies were negatively correlated with left ventricular ejection fraction (LVEF), while positively with N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with DCM. CONCLUSIONS: Circulating activated MDSCs might play significant immunomodulatory roles in the pathogenesis of DCM.

Reversible SAHH inhibitor ameliorates MIA-induced osteoarthritis of rats through suppressing MEK/ERK pathway.

Osteoarthritis (OA) is characterized by gradual articular cartilage degradation, accompanied by persistent low-grade joint inflammation, correlating with radiographic and pain-related progression. The latent therapeutic potential of DZ2002, a reversible inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH), holds promise for OA intervention. This study endeavored to examine the therapeutic efficacy of DZ2002 within the milieu of OA. The cytotoxicity of DZ2002 was evaluated using the MTT assay on bone marrow-derived macrophages. The inhibitory impact of DZ2002 during the process of osteoclastogenesis was assessed using TRAP staining, analysis of bone resorption pits, and F-actin ring formation. Mechanistic insights were derived from qPCR and Western blot analyses. Through the intra-articular injection of monosodium iodoacetate (MIA), an experimental rat model of OA was successfully instituted. This was subsequently accompanied by a series of assessments including Von Frey filament testing, analysis of weight-bearing behaviors, and micro-CT imaging, all aimed at assessing the effectiveness of DZ2002. The findings emphasized the effectiveness of DZ2002 in mitigating osteoclastogenesis induced by M-CSF/RANKL, evident through a reduction in TRAP-positive OCs and bone resorption. Moreover, DZ2002 modulated bone resorption-associated gene and protein expression (CTSK, CTR, Integrin ß3) via the MEK/ERK pathway. Encouragingly, DZ2002 also alleviates MIA-induced pain, cartilage degradation, and bone loss. In conclusion, DZ2002 emerges as a potential therapeutic contender for OA, as evidenced by its capacity to hinder in vitro M-CSF/RANKL-induced osteoclastogenesis and mitigate in vivo osteoarthritis progression. This newfound perspective provides substantial support for considering DZ2002 as a compelling agent for osteoarthritis intervention.

Large-scale sequencing of normalized full-length cDNA library of soybean seed at different developmental stages and analysis of the gene expression profiles based on ESTs.

Although GenBank has now covered over 1,400,000 expressed sequence tags (ESTs) from soybean, most ESTs available to the public have been derived from tissues or environmental conditions rather than developing seeds. It is absolutely necessary for annotating the molecular mechanisms of soybean seed development to analyze completely the gene expression profiles of its immature seed at various stages. Here we have constructed a full-length-enriched cDNA library comprised of a total of 45,408 cDNA clones which cover various stages of soybean seed development. Furthermore, we have sequenced from 5' ends of these clones, 36,656 ESTs were obtained in the present study. These EST sequences could be categorized into 27,982 unigenes, including 22,867 contigs and 5,115 singletons, among which 27,931 could be mapped onto soybean 20 chromosome sequences. Comparative genomic analysis with other plants has revealed that these unigenes include lots of candidate genes specific to dicot, legume and soybean. Approximately 1,789 of these unigenes currently show no homology to known soybean sequences, suggesting that many represent mRNAs specifically expressed in seeds. Novel abundant genes involved in the oil synthesis have been found in this study, may serve as a valuable resource for soybean seed improvement.

Gene expression profiling of Sinapis alba leaves under drought stress and rewatering growth conditions with Illumina deep sequencing.

Sinapis alba has many desirable agronomic traits including tolerance to drought. In this investigation, we performed the genome-wide transcriptional profiling of S. alba leaves under drought stress and rewatering growth conditions in an attempt to identify candidate genes involved in drought tolerance, using the Illumina deep sequencing technology. The comparative analysis revealed numerous changes in gene expression level attributable to the drought stress, which resulted in the down-regulation of 309 genes and the up-regulation of 248 genes. Gene ontology analysis revealed that the differentially expressed genes were mainly involved in cell division and catalytic and metabolic processes. Our results provide useful information for further analyses of the drought stress tolerance in Sinapis, and will facilitate molecular breeding for Brassica crop plants.

Analysis of the differential expression of the genes related to Brassica napus seed development.

To screen the genes related to Brassica napus seed development at pattern formation and maturation stages, the suppression subtractive cDNA libraries of B. napus cultivar Zhongshuang 6 were constructed with its embryos at 10 days after flowering (10 DAF) and 30 days after flowering (30 DAF) through suppression subtractive hybridization (SSH) technology. The positive clones were screened by PCR and dot blot hybridization, and then sequenced. High quality expressed sequence tags (ESTs) were used for COG functional classification with COGNITOR software, as well as analysis and annotation with BLAST software. Tissue-specific detection of five genes screened was performed by RT-PCR in root, stem, leaf, flower, bud, pod, and embryo tissues. The insert size ranged from 100 to 900 bp, with an average size of about 500 bp. According to COG functional classification database, the differentially expressed genes mainly involved in lipid and amino acid metabolism, signal transduction, post-transcriptional modification, and etc. The results from RT-PCR detection of the five differentially expressed genes indicated that genes 2-96 and 2-352 presented embryo-specific expression, gene 1-385 expressed in parts of tissues, and genes 1-71 and 1-682 expressed in all tissues. Two genes were found to be involved in seed development, lipid and protein metabolisms, two genes may be involved in signal transduction, one gene could not match with the homologous sequences known to date and was likely a new gene. These results are helpful for future gene cloning and their functional analysis.

Effect of patchouli alcohol on Helicobacter pylori-induced neutrophil recruitment and activation.

Neutrophil infiltration typically occurs in Helicobacter pylori (H. pylori)-induced acute gastritis; however, this immune response fails to eradicate H. pylori in vivo. Moreover, reactive oxygen species (ROS), which are generated by neutrophils, cause severe damage to gastric mucosa. Patchouli alcohol (PA) has been reported to have effective anti-oxidative and anti-H. pylori activities, and we investigated its effects on H. pylori-induced neutrophil recruitment and activation in this research. In neutrophil recruitment experiment, H. pylori was injected into rat air pouch to explore the effects of PA (10, 20 and 40 mg/kg) on acute inflammatory response. The results revealed that PA significantly reduced the weight of exudate and the number of neutrophils in the air pouch. Meanwhile, remarkable decrements in TNF-α and IL-8 levels in exudates were observed. In neutrophil activation experiment, rat neutrophils were isolated and activated by using 50 µg/mL H. pylori water-soluble surface protein with or without the treatment of PA (5, 10 or 20 µmol/L). Results indicated that PA not only significantly inhibited the production of ROS, but also reduced the gene and protein expressions of p22/p47-phoxes, and the binding of p22/p47-phoxes. Furthermore, the influence of PA on the neutrophil activation genes of H. pylori (h-nap and sabA) was investigated, and the results showed that expressions of h-nap and sabA were remarkably decreased after PA treatment. In conclusion, PA reduced the recruitment and activation of neutrophils induced by H. pylori, as shown by its inhibition of pro-inflammatory factor generation, p22/p47-phoxes function and H. pylori neutrophil activation-related gene expression.

An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid.

Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKß/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products.

[Laws of the change in genotype entropy from F1 to Fn for pairs of independent genes of the selfing population].

This paper constructs a mathematic model for the change in each generation's genotype entropy of the selfing population with independent heterogenes, and describes a ternary tree algorithm to compute the proportion of every genotype. It reveals a linear relationship between the population genotype entropy and the pairs of independent heterogenes m, and a nonlinear relationship between the population genotype entropy and the ordinal number n of a selfing generation. As n is fixed, the population genotype entropy with m pairs of independent heterogenes is m times as many as that with only one pair. As the pairs of the independent heterogenes m is fixed, the population genotype entropy increases generation after generation from F1 to F3, reaching the maximum value at F3, and decreases generation after generation from F3, reaching equilibrium finally at the generation whose genotype entropy is minimum. In this paper, the significance of crossbreeding is also discussed.

Construction of a high-quality genomic BAC library for Chinese peanut cultivar Zhonghua 8 with high oil content.

BACKGROUND: Arachis hypogaea L. (2n = 4× = 40, AABB) is one of the most important oil and economic crop plants in the word. This species has the largest genome size of about 2,813 Mb among the oil crop species. Zhonghua 8 is a peanut cultivar planted widely in central China and has several superior traits including high oil content, high yield and disease resistance. A high-quality BAC library of Zhonghua 8 was constructed for future researches on the genomics of Chinese peanut cultivars. RESULTS: A Hin d III-digested genomic BAC (bacterial artificial chromosome) library was constructed with the genomic DNA from leaves of Zhonghua 8. This BAC library consists of 160,512 clones and the average insert is estimated about 102 kb ranging from 30 to 150 kb. The library represents about 5.55× haploid genome equivalents, and provides a 99.71% probability of finding specific genes. The empty-vector rate is under 5 percent detected from 200 randomly selected clones. Probing of 384 clones with the psbA gene of barley chloroplast and the atp6 gene of rice mitochondrion indicated that the contamination with organellar DNA is insignificant. Successive subculture of three clones showed that the inserts are stable in one hundred generations. CONCLUSIONS: This study presented the construction of a high-quality BAC library for the genome of Chinese cultivated peanut. Many essential experiences were summarized in the present study. This BAC library can serve as a substantial platform for development of molecular marker, isolation of genes and further genome research.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp.

BACKGROUND: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. METHODS: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4 degrees C. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 microg/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. RESULTS: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5.38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. CONCLUSIONS: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus).

Genomic in situ hybridization analysis for identification of introgressed segments in alloplasmic lines from Zea mays x Zea diploperennis.

In this study, the tested four alloplasmic inbred lines, a2-4, a2-5, b1-1 and b2-1 were propagated from the same disease resistant individual in the parthenogenetic progenies of Zea mays L. cv. Lu 9 x Zea diploperennis (DP). All the lines except a2-5 were resistant to Helminthosporium turcium Pass and H. maydis Nisik. Introgressed DP segments in these lines were detected by both Southern hybridization and genomic in situ hybridization (GISH). The results of Southern hybridization showed that DP species-specific DNA sequences had been introgressed into the genomes of alloplasmic lines. The Southern hybridization band patterns in all of the tested lines were consistent with those of DP. Genomic in situ hybridization (GISH) signals were detected on 7 different chromosome pairs in lines a2-4 and a2-5, on 5 chromosome pairs in b1-1 and on 4 chromosome pairs in b2-1. The features of introgression, and disease resistant genes in the introgressed segments, as well as the gene silence or elimination in some alloplasmic lines are discussed.