Aloperine alleviates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome activation.

RATIONALE: Excessive activation of the NLRP3 inflammasome is involved in the pathological progression of acute lung injury (ALI). Aloperine (Alo) has anti-inflammatory effects in many inflammatory disease models; however, its role in ALI remains elusive. In this study, we addressed the role of Alo in NLRP3 inflammasome activation in both ALI mice and LPS-treated RAW264.7 cells. METHODS: The activation of the NLRP3 inflammasome in LPS-induced ALI lungs was investigated in C57BL/6 mice. Alo was administered in order to study its effect on NLRP3 inflammasome activation in ALI. RAW264.7 cells were used to evaluate the underlying mechanism of Alo in the activation of the NLRP3 inflammasome in vitro. RESULTS: The activation of the NLRP3 inflammasome occurs in the lungs and RAW264.7 cells under LPS stress. Alo attenuated the pathological injury of lung tissue as well as downregulates the mRNA expression of NLRP3 and pro-caspase-1 in ALI mice and LPS-stressed RAW264.7 cells. The expression of NLRP3, pro-caspase-1, and caspase-1 p10 were also significantly suppressed by Alo in vivo and in vitro. Furthermore, Alo decreased IL-1ß and IL-18 release in ALI mice and LPS-induced RAW264.7 cells. In addition, ML385, a Nrf2 inhibitor, weakened the activity of Alo, which inhibited the activation of the NLRP3 inflammasome in vitro. CONCLUSION: Alo reduces NLRP3 inflammasome activation via the Nrf2 pathway in ALI mice.

Isolation and Characterization of Ftsz Genes in Cassava.

The filamenting temperature-sensitive Z proteins (FtsZs) play an important role in plastid division. In this study, three FtsZ genes were isolated from the cassava genome, and named MeFtsZ1, MeFtsZ2-1, and MeFtsZ2-2, respectively. Based on phylogeny, the MeFtsZs were classified into two groups (FtsZ1 and FtsZ2). MeFtsZ1 with a putative signal peptide at N-terminal, has six exons, and is classed to FtsZ1 clade. MeFtsZ2-1 and MeFtsZ2-2 without a putative signal peptide, have seven exons, and are classed to FtsZ2 clade. Subcellular localization found that all the three MeFtsZs could locate in chloroplasts and form a ring in chloroplastids. Structure analysis found that all MeFtsZ proteins contain a conserved guanosine triphosphatase (GTPase) domain in favor of generate contractile force for cassava plastid division. The expression profiles of MeFtsZ genes by quantitative reverse transcription-PCR (qRT-PCR) analysis in photosynthetic and non-photosynthetic tissues found that all of the MeFtsZ genes had higher expression levels in photosynthetic tissues, especially in younger leaves, and lower expression levels in the non-photosynthetic tissues. During cassava storage root development, the expressions of MeFtsZ2-1 and MeFtsZ2-2 were comparatively higher than MeFtsZ1. The transformed Arabidopsis of MeFtsZ2-1 and MeFtsZ2-2 contained abnormally shape, fewer number, and larger volume chloroplasts. Phytohormones were involved in regulating the expressions of MeFtsZ genes. Therefore, we deduced that all of the MeFtsZs play an important role in chloroplast division, and that MeFtsZ2 (2-1, 2-2) might be involved in amyloplast division and regulated by phytohormones during cassava storage root development.

Identification, Expression, and Functional Analysis of the Fructokinase Gene Family in Cassava.

Fructokinase (FRK) proteins play important roles in catalyzing fructose phosphorylation and participate in the carbohydrate metabolism of storage organs in plants. To investigate the roles of FRKs in cassava tuber root development, seven FRK genes (MeFRK1-7) were identified, and MeFRK1-6 were isolated. Phylogenetic analysis revealed that the MeFRK family genes can be divided into α (MeFRK1, 2, 6, 7) and ß (MeFRK3, 4, 5) groups. All the MeFRK proteins have typical conserved regions and substrate binding residues similar to those of the FRKs. The overall predicted three-dimensional structures of MeFRK1-6 were similar, folding into a catalytic domain and a ß-sheet ''lid" region, forming a substrate binding cleft, which contains many residues involved in the binding to fructose. The gene and the predicted three-dimensional structures of MeFRK3 and MeFRK4 were the most similar. MeFRK1-6 displayed different expression patterns across different tissues, including leaves, stems, tuber roots, flowers, and fruits. In tuber roots, the expressions of MeFRK3 and MeFRK4 were much higher compared to those of the other genes. Notably, the expression of MeFRK3 and MeFRK4 as well as the enzymatic activity of FRK were higher at the initial and early expanding tuber stages and were lower at the later expanding and mature tuber stages. The FRK activity of MeFRK3 and MeFRK4 was identified by the functional complementation of triple mutant yeast cells that were unable to phosphorylate either glucose or fructose. The gene expression and enzymatic activity of MeFRK3 and MeFRK4 suggest that they might be the main enzymes in fructose phosphorylation for regulating the formation of tuber roots and starch accumulation at the tuber root initial and expanding stages.

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava.

Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven HXK genes (MeHXK1-7) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2-7) based on their N-terminal sequences. MeHXK1-5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6-7 lacked some of the conserved regions. An expression analysis of the MeHXK genes in cassava organs or tissues demonstrated that MeHXK2 is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of MeHXK2 and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.

Isopentenyl transferase gene (ipt) downstream transcriptionally fused with gene expression improves the growth of transgenic plants.

This research reports a promising approach to increase a plant's physiological cytokinin content. This approach also enables the increase to play a role in plant growth and development by introducing the ipt gene to downstream transcriptionally fuse with other genes under the control of a CaMV35S promoter, in which the ipt gene is far from the 35S promoter. According to Kozak's ribosome screening model, expression of the ipt gene is reduced by the terminal codon of the first gene and the internal untranslated nucleotides between the fused genes. In the transgenic plants pVKH35S-GUS-ipt, pVKH35S-AOC-ipt, and pVKH35S-AtGolS2-ipt, cytokinins were increased only two to threefold, and the plants grew more vigorously than the pVKH35S-AOC or pVKH35S-AtGolS2 transgenic plants lacking the ipt gene. The vigorous growth was reflected in rapid plant growth, a longer flowering period, a greater number of flowers, more seed product, and increased chlorophyll synthesis. The AOC and AtGolS2 genes play a role in a plant's tolerance of salt or cold, respectively. When the ipt gene transcriptionally fuses with AOC or AtGolS2 in the frame of AOC-ipt and AtGolS2-ipt, slight cytokinin increases were obtained in their transgenic plants; furthermore, those increases played a positive role in improvements of plant growth. Notably, an increased cytokinin volume at the physiological level, in concert with AtGolS2 expression, enhances a plant's tolerance to cold.

CaCl2 enhanced somatic embryogenesis in Manihot esculenta Crantz.

Primary cassava somatic embryos were induced on a medium without CaCl(2), however, no or only a few secondary somatic embryos were formed from them. With 15 mM CaCl(2) in the medium for induction of cassava primary embryos, more secondary somatic embryos were produced from them, and they were much effective in maintaining their embryogenic capacity than the controls of embryos which were induced without CaCl(2).

Embryo and anther regulation of the mabinlin II sweet protein gene in Capparis masaikai Lévl.

Mabinlin II is one of the major sweet proteins stored in the seeds of Capparis masaikai Lévl. Its promoter region (779 bp) located 5' upstream of the mabinlin II gene has been isolated and named as MBL-779 (GenBank accession number, EU014073). This promoter contains two typical TATA box regions and a series of motifs related to seed-specific promoters, such as ACGT motifs, RY motif, napin motif, and G box. The MBL-779 promoter drove GUS gene to transiently express in the embryos of bean, maize, and rice seeds or to constantly express in the embryos and anthers of the transgenic Arabidopsis. The MBL-779 promoter regulated gene expression from approximately the 12th day and peaked on approximately the 16th day after flowering in Arabidopsis. The -300-bp promoter region is a minimal sequence required to functionally regulate gene expression. The CAATs at -325 to -322 bp and -419 to -416 bp and the region at -485 to -770 bp play a role in the quantitative regulation of gene expression. The RY motif, CATGAC, at -117 to -112 bp and the ACGT within the G box (CACGTG) at -126 to -123 bp positively regulate gene expression.

Activation of genes presumed in cytokinin signal pathway of two-component system in response to the increased cytokinin contents in ipt-GUS transgenic Arabidopsis.

The ipt-GUS activated transgenic Arabidopsis had 20-25 fold higher cytokinin contents than the wild type. Changes in cytokinin content in vivo triggering gene expressions involved in signal pathway of two-component system have been analyzed on the day of 6 d, 12 d, 20 d and 30 d after seed cultivation on MS medium in light conditions. The results showed that the two cytokinin receptors, His-kinase CRE1 was more sensitive to the increased cytokinin contents than CKI1. Arabidopsis response regulators, ARR4 and ARR5, were induced by the increased cytokinin contents at different time after seed germination. ARR4 responded to cytokinins at early time of seed germination, especially on the 6 d when seedlings were around true leaf initiation, while cytokinins induced ARR5 activation after 6 d of seed cultivation in light conditions, an obvious increase was on the 20 d when seedlings were around inflorescent shoot initiation. Hpt-type transmitter kinase AHP4 increased its activation by cytokinin induction only between the 20 d and 30 d after seed cultivation in light conditions, and an obvious increase was on the 20 d.

A new amide from Asarum forbesii Maxim.

A highly unsaturated new amide, (2E,4Z,8Z,10Z)-N-isobutyl-2,4,8,10-dodecatetraenamide (1), was isolated in very small quantities from the whole plant of Asarum forbesii Maxim. together with four known compounds, (2E,4E,8Z,10E)-N-isobutyl-2,4,8,10-dodecatetraenamide (2), (-)-sesamin (3), (-)-asarinin (4) and (E)-asarone (5). The Z/E isomers, 1 and 2, were separated successfully by developed silver-ion medium-pressure liquid chromatography (SIMPLC). Compound 2 and the two diastereoisomers, 3 and 4, were isolated from this plant for the first time. The characterization of these compounds was achieved by various spectroscopic methods.

[Study on GC fingerprint of the constituents in Herba Asari].

OBJECTIVE: To establish a method for GC fingerprint determination of the chemical constituents in Herba Asari. METHOD: GC and GC-MS were used to optimize the fingerprint determination method, and identify the main peaks in the GC fingerprint. RESULT: A preferable method for GC fingerprint determination of the chemical constituents in Herba Asari was established. CONCLUSION: A general acquaintance of the chemical constituents in Herba Asari can be obtained by using the preferable GC fingerprint determination method, which is useful for quality evaluation of the crude drug of Herba Asari.