[Efficacy of shenfu injection as adjuvant therapy in treating patients of ischemic cardiomyopathy with heart insufficiency].

OBJECTIVE: To evaluate the clinical efficacy of Shenfu Injection (SFI), as a adjuvant therapy, in treating patients of ischemic cardiomyopathy with heart insufficiency (ICP-HI). METHODS: One hundred patients of ICP-HF were equally randomized into two groups, the SFI group and the control group. All received the conventional treatment, but to patients in the SFI group SFI was given additionally via intravenous injection, 60 mL once a day, 10 days each month, the treatment course was 6 months. Changes of cardial functional grading, 6-min walking distance, echocardiographic indices, plasma N terminal pro-brain natriuretic peptide (pro-BNP) level were observed before and after treatment, and the occurrence of major adverse cardiovascular events (MACE) and mortality in patients were observed as well. RESULTS: As compared with the conventional treatment alone, additional application of SFI showed a more significant efficacy in improving NYHA functional grade and 6-min walking distance, reducing the diameters of left ventricular at end diastole and systole, increasing left ventricular ejection fraction, and decreasing plasma N terminal pro-BNP level (P <0.05). The occurrence of MACE and the mortality in the SFI group were significantly lower than those in the control group respectively (P <0.05). CONCLUSIONS: Based on the conventional treatment, the adjuvant therapy of SFI could improve the cardiac function, improve the quality of life, ameliorate ventricular reconstruction, and decrease the occurrence of cardiovascular events in patients of ICP-HI.

MicroRNA-21 Mediates Angiotensin II-Induced Liver Fibrosis by Activating NLRP3 Inflammasome/IL-1ß Axis via Targeting Smad7 and Spry1.

AIMS: Angiotensin II (AngII), a vasoconstrictive peptide of the renin-angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1-7) [Ang-(1-7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1ß axis in liver fibrosis. RESULTS: In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1-7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1-7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects. Innovation and Conclusions: Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1-7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1-20.

Functional expression of the cotton gibberellic acid oxidase homologous gene GhGA20ox1 in tobacco.

To determine the physiological function of GhGA20ox1, a homologous gene of GA 20-oxidase from elongating cotton fibers, we expressed this gene ectopically in Nicotiana benthamiana. Reverse transcription-PCR analysis showed that the GhGA20ox1 gene was expressed in the transgenic plants at various levels. It was demonstrated that overexpression of GhGA20ox1 enhanced preferentially the GA(4+7) biosynthesis in N. benthamiana and conferred GA-overproduction characters to transformants. The extent of phenotypic alteration in the transgenic plants was found to correlate with the transcriptional levels of GhGA20ox1 and GA contents. Results indicated that the GhGA20ox1 gene promoted the biosynthesis of the active GAs (GA(4+7)) in transgenic tobacco plants therefore represents a useful gene for manipulating GA levels.

Inhibition of alpha-synuclein fibrillization by dopamine analogs via reaction with the amino groups of alpha-synuclein. Implication for dopaminergic neurodegeneration.

Fibrillization of alpha-synuclein (alpha-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit alpha-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of alpha-Syn chain, generating alpha-Syn-quinone adducts. It is likely that the amino groups of alpha-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked alpha-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact alpha-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of alpha-Syn with DA is relevant to the selective dopaminergic loss in PD.

An unstructured region is required by GAV homologue for the fibrillization of host proteins.

Accumulating evidence shows that some amyloidogenic proteins contain core sequences, which are critical for their fibrillization. Core sequences of alpha-synuclein, beta-amyloid peptide and prion protein usually reside in their unfolded regions and share a conserved consensus (VGGAVVAGV) designated as GAV homologue. Here we investigate the role of unfolded regions in fibrillization after GAV homologue is attached to the C-terminus or inserted into the loop regions of different host proteins, namely alpha -Syn1-65, gamma-synuclein, E. coli thioredoxin and immunoglobulin G binding B1 domain of streptococcal protein G. The results imply that an unstructured region is required by GAV homologue for the fibrillization of host proteins. A number of amyloidogenic proteins with core sequences located in unstructured regions are summarized and discussed in details. The finding may provide further insight into the elucidating of the molecular mechanism underlying the fibrillization of alpha-Syn, Abeta and PrP as well as other amyloidogenic proteins.

[Cloning and expression analysis of two Rac genes from cotton (Gossypium hirsutum L.)].

Plant Rac proteins belong to an important group of signal switches anchoring on membranes, involved in various physiological processes including cell polar growth, synthesis of secondary wall, resistance response and hormone signaling. In the attempt to elucidate the molecular mechanism of initiation and elongation of cotton fiber, two cotton Rac protein genes, designated as GhRacA and GhRacB, were amplified from elongating fibers and cloned. It was demonstrated that, the cDNA of GhRacA contained 959 bp and encoded a putative polypepetide of 211 aa, while GhRacB was 920 bp in length, encoding a predicted protein of 195 aa. These two cotton Rac proteins, GhRacA and GhRacB, contained conserved regions involved in GTP/GDP binding and activation, an effector region and a polybasic region. A conserved prenylation site CSIL was found in GhRacB, while no apparent prenylation site was discovered in GhRacA. Sequence comparisons showed that GhRacA and GhRacB were two novel Rac proteins from cotton. The expression patterns of GhRacA and GhRacB was analyzed by RT-PCR. It was demonstrated that these two Rac protein genes were both expressed in root, hypocotyls, stem, leaf and fibers, and the highest level of transcripts was to accumulate in the fibers at the stage of initiation and elongation, suggesting that the two Rac genes, GhRacA and GhRacB, might play an important role in the early stage of fiber development.

[The cDNA-AFLP differential display in developing anthers between cotton male sterile and fertile line of "Dong A"].

cDNA-AFLP, an effective method for mRNA differential display, was employed to compare the gene expression in developing anthers between the male sterile and fertile plants of cotton (Gossypium hirsutum L.) cultivar, Dong A. In the micro-spore stage, there were more differential bands of cDNA-AFLP than that in the meio-phase stage. Among 64 differential fragments produced by cDNA-AFLP, three were randomly selected for further analysis. RNA dot blotting showed that the GHA27 transcript was expressed mainly in floral tissues; on the other hand, the GHA28 and GHA47 transcripts were present specifically in anther. BLAST analysis demonstrated that GHA27 was highly similar to the plant ADP-ribosylation factor genes, while GHA28 and GHA27 were shown no significant similar to any sequences in the available databases.

[Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development.

[Cloning and characterization of a LRR resistance like (GhLRR-RL) protein gene from cotton (Gossypium hirsutum L.)].

LRR (Leucine rich repeat) proteins are involved in various biological processes. RACE was used to extend the 5' and 3' unknown sequence of a cDNA-AFLP fragment from cotton ovule, which had sequence similarity to the Arabidopsis thaliana LRR resistance-like (LRR-RL) protein. By joining the sequence of cDNA-AFLP fragment and its 3' and 5' RACE product, it was found that the full-length cDNA (GenBank Accession No. AY040532) of 1259 bp contained an ORF of 987 bp and a putative poly(A) signal. The deduced polypeptide contained 328 amino acids, and consisted of 10 LRRs with extracytoplasmic LRR consensus LXXLXXLXXLXLXXNXGXIPXX. It was demonstrated that the LRR-RL proteins of cotton and A. thaliana were highly homologous, with a distinct structure from other plant LRR proteins, suggesting that LRR-RL proteins belonged to a new LRR protein family. Using 3' RACE, the expression of LRR-RL gene was detected in root, hypocotyls, fiber, and ovules in various developmental stages.

[Cloning of a MADS box protein gene (GhMADS1) from cotton (Gossypium hirsutum L.)].

As a kind of transcription factors, MADS-box protein plays an important role in various cellular processes, especially in the development of floral organs. Based on the contig analysis of the cotton ESTs, the coding region of a cotton MADS-box protein (GhMADS1) was obtained by RT-PCR from floral buds of cotton (G. hirsutum). The cloned fragment of 713 bp (GhMADS1, GenBank accession no. AF538965) contains an open reading frame of 711 bp,coding a polypeptide of 236 amino acids. It was demonstrated that the deduced GhMADS1 protein was highly homologous to the AGL2 group of MADS-box proteins from Vitis vinifera, Nicotiana sylvestris, Petunia hybrida, Arabidopsis thaliana and Antirrhinum majus. Phylogenetic analysis also indicated that GhMADS1 belongs to the AGL2 group of MADS-box proteins. RT-PCR analysis showed that GhMADS1 gene expressed in petals, stamens, ovules and fibers, but not in roots, stems, leaves, bracts and sepals. The strongest expression of GhMADS1 gene was detected in petals. But in floral buds of a cotton homeotic mutant (CHV1), whose floral organs are all converted to bract leaf-like organs, the transcript of GhMADS1 gene was not detected. It was proposed that GhMADS1 gene would be crucial to the development of cotton floral organs.

[Prediction of yield and yield components in hybrid rice by using molecular markers].

Yield and yield components in hybrid rice were investigated using AFLP, RAPD and SSR markers. Ten restorer and five male-sterile lines were crossed in all possible pairs resulting in 50 crosses. Positive loci, effect-increasing loci, effect-decreasing loci and non-environmental loci were selected from the 931 marker loci surveyed in the 15 parental lines and their correlation with yield and yield components were analyzed. The results indicated as follows (1) The correlation between genetic difference and yield and yield components calculated on all three molecular loci failed to reach significant level for most of the traits investigated and can not be used to predict yield and yield components directly. (2) Positive loci were of limited usefulness in the prediction of yield and yield components for their variation with different traits investigated despite that they can improve the correlation coefficient in some degree. (3) Effect-increasing and effect-decreasing loci can greatly improve correlation coefficient and may be used to predict the yield and yield components for their consistence with the environment. (4) The coefficient based on non-environmental loci was high though it was a bit lower than that based on effect-increasing and effect-decreasing loci. It indicated that environment had great effect on yield and yield components in rice.

[Cloning and characterization of the PTS2 receptor gene (GhPex7) from cotton (Gossypium hirsutum L.)].

Peroxisomal targeting signals (PTS), including PTS1 and PTS2, were proposed to play an important role in introducing proteins into the peroxisomal matrix. Pex7, the PTS2 receptor, is crucial to the import of PTS2 proteins. Based on the sequence of a fragment (F010) recovered from cDNA-AFLP, the full-length cDNA sequence was obtained by RACE and the contig in cotton ESTs, and the coding sequence was further cloned. The GhPex7 cDNA, 1314 bp in length, contained 5 non-coding (77 bp) upstream, 3 complete downstream with polyA signal tail and ORF of 954 bp, which coded for a deduced protein of 317 amino acids. The deduced protein had a predicted MW 35.57 kDa and a pI of 5.603. Homology analysis demonstrated that GhPex7 protein contained three highly conserved domains and three G-bata domains of WD-40 proteins family. The sequence of nucleotides and amino acids shared 83% and 76% identity with known Arabidopsis AtPex7, respectively, and its amino acid sequence shared from 28% to 42% identity with those of Drosophila melanogaster, Saccach, cerevisiae, Mus musculus and Homo sapiens. Southern blotting suggested that at least two copies of GhPex7 gene existed in Gossypium hirstum genome. By Northern blotting and RT-PCR analysis, expression of the GhPex7 was detected in roots, stems, leaves, buds, ovules and fibers, however it was stronger in leaves and stems than in other tissues. At the stage of cotton's ovules and fibers development, RT-PCR analysis also indicated that expression activity of GhPex7 in ovule at 0DPA mutant plant (no-fiber) was stronger than in wild type plant, its expression in wild type fiber at 23DPA was stronger than at 12, 16 DPA too.

[PCR walking in cotton genome using YADE method].

A Y-shaped adaptor dependent extension (YADE) method was developed to amplify the unknown genomic sequences adjacent to a known sequence, with the single-primer amplification completely suppressed. Using this method, two fragments, F027S and F027A, adjacent to a cotton ovule cDNA fragment (F027), were amplified using genomic DNA. It was demonstrated that F027S and F027A had 104 bp and 175 bp overlapped to F027, and contained 1 and 3 putative introns, respectively, all of which included conserved border sequence GT-AG and a putative branch site. The possible improvements and applications of YADE method are further discussed.

[Cloning and characterization of a homologous gene of plant class V chitinase from balsampear, Momordica charantia Linn].

Balsampear (M. charantia Linn.) is a vegetable crop, highly resistant to pathogens. Chitinases were proposed to play an important role in the defense response of this crop. Based on the N-terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tobacco class V chitinase gene, was amplified from the leaf RNA using 3'RACE, and the corresponding 5' sequence was further amplified by the Y-RACE method. By joining the two amplified fragments, the full-length cDNA of M. charatica homologous gene of plant class V chitinase (McChi5) was obtained. The 1348 bp cDNA contained an ORF of 1044 bp, which coded for a polypeptide of 347 amino acids. The deduced polypeptide had a predicted molecular weight of 38.3 kD and a pI of 5.77. Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tobacco class V chitinases, several putative chitinases and chitinase-like proteins of Arabidopsis thiliania, and some chitinases from mammals, insects and bacteria. Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M. charatica genome. By RNA dot blotting analysis, expression of the McChi5 gene was detected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment. The biological functions and the potential applications of Mochi5 gene were discussed.

Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis.

Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.

Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants.

A balsam pear (Momordica charantia L.) chitinase (Mcchit1) was purified and sequenced at the N-terminal. The genomic and cDNA coding sequences of Mcchit1 were cloned by rapid amplification of 3' cDNA ends (3'-RACE) and the Y-shaped adaptor dependent extension (YADE) method. Sequence analysis showed that the Mcchit1 protein is a class I chitinase containing a chitin-binding domain and a catalytic domain, but no C-terminal extension. Northern blot indicated that the Mcchit1 transcription is wound-inducible. Overexpression of Mcchit1 dramatically increased intercellular and intracellular endochitinase activities, suggesting that the Mcchit1 gene encodes a secretory endochitinase. It was also found that overexpression of Mcchit1 significantly enhanced resistance to the plant pathogenic fungus Phytophthora nicotianae in transgenic N. benthamiana plants and against Verticillium wilt in transgenic cottons, indicating that the Mcchit1 gene can be a useful gene in plant engineering against fungal diseases.

A peptide motif consisting of glycine, alanine, and valine is required for the fibrillization and cytotoxicity of human alpha-synuclein.

Amyloid-like aggregation or fibrillization of alpha-synuclein (alpha-Syn) and the filamentous deposits in Lewy bodies are believed to be closely associated with several fatal neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease. Here, we report the importance of a nine-residue peptide motif, (66)VGGAVVTGV(74), in the fibrillization and cytotoxicity of human alpha-Syn. Mutagenesis combined with thioflavin T fluorescence detection, atomic force microscopic imaging, and cytotoxicity assays reveal that deletion of this sequence completely eliminates alpha-Syn fibrillization and cell toxicity. However, deletion of the (71)VTGV(74) sequence decreases the fibrillization rate while the cytotoxicity remains unchanged. Incorporation of charged residues within this region slows aggregation and even impedes filament formation. In addition, substitution of Gly68 with Ala or C-terminal truncations of alpha-Syn accelerate the fibrillization processes. Circular dichroism studies suggest that beta-sheet formation is often concomitant with filament formation. Thus, this segment, namely, the GAV motif, is responsible for aggregation or fibrillization of alpha-Syn and perhaps other amyloidogenic proteins. The oligomers formed during fibrillogenesis might be associated with the cytotoxicities of various alpha-Syn species. This finding may provide further insight into the understanding of the molecular mechanism underlying the fibrillogenesis implicated in neurodegeneration as well as aid in drug design and development of transgenic models.