RP1, a RAGE antagonist peptide, can improve memory impairment and reduce Aß plaque load in the APP/PS1 mouse model of Alzheimer's disease.

Amyloid-ß (Aß) accumulation is a pathological hallmark of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is involved in the production and accumulation of Aß. RP1, a peptide antagonist of RAGE, was screened by phage display technology in our previous studies, and its neuroprotective effects on an AD cell model have been confirmed. However, its efficacy in vivo remains unclear. Here, the intranasal delivery of RP1 to APPSwe/PS1dE9 (APP/PS1) mice significantly improved memory impairment and relieved the Aß burden by decreasing the expression of amyloid precursor protein and ß-secretase. RNA-sequencing (RNA-seq) was utilized to identify differentially expressed genes (DEGs) in APP/PS1 mice after RP1 administration. Several DEGs in RAGE downstream signalling pathways were downregulated. Some transcription factors (such as Fos) and the pathways enriched in the remarkable modules may also be related to the efficacy of RP1. In conclusion, RP1 significantly improves the AD symptoms of APP/PS1 mice, and the RNA-seq results provide new ideas for elucidating the possible mechanisms of RP1 treatment.

Alanylglutamine Relieved Asthma Symptoms by Regulating Gut Microbiota and the Derived Metabolites in Mice.

OBJECTIVE: Allergic asthma is a chronic inflammatory disease, which seriously affects the life quality of patients, especially children. Alanylglutamine is a nutritional supplement with potential protective and anti-inflammatory effects, but its function in allergic asthma remains elusive. In this study, we focused on the investigations of the roles and functional mechanism of Alanylglutamine in asthma. METHODS: Ovalbumin (OVA) induction was utilized to establish a mouse asthma model. 16S rDNA sequencing was performed to compare the diversity of intestinal microorganisms under different treatments. Gas chromatography was utilized to screen the intestinal microbe-short-chain fatty acids in the stool. The lung tissue was extracted to determine signaling pathways, including AMPK, NF-κB, mTOR, STAT3, IKKß, TGF-ß, and IL-1ß through Western blot or RT-qPCR. RESULTS: It was observed that Alanylglutamine reduced the cytokine in OVA-induced allergic asthma mice. H&E staining showed obvious pneumonia symptoms in the asthma group, while Alanylglutamine alleviated the inflammatory infiltration. Alanylglutamine reversed gut microbiota compositions in OVA-induced allergic asthma mice and enhanced the butyric acid level. The protective role of Alanylglutamine may be associated with the gut microbiota-butyric acid-GPR43 pathway in asthma mice. In contrast to the OVA group, Alanylglutamine activated the protein expression of P-AMPK/AMPK and inhibited the protein expression of P-mTOR/mTOR, P-P65/P65, P-STAT3/STAT3, P-IKKß/IKKß, TGF-ß, and IL-1ß, with similar effects from butyric acid. CONCLUSION: The results indicated that Alanylglutamine might be beneficial for asthma, and its effect was achieved through the regulation on microbiota and the derived metabolites. The therapeutic effects might be associated with AMPK, NF-κB, mTOR, and STAT3 signaling pathways. These findings will help identify effective therapeutic direction to alleviate allergic inflammation of the lungs and airways.

How Do Structurally Distinct Compounds Exert Functionally Identical Effects in Combating Obesity?

Although the concept of inflammatory obesity remains to be widely accepted, a plethora of antibiotics, anti-inflammatory agents, mitochondrial uncouplers, and other structurally distinct compounds with unknown mechanisms have been demonstrated to exert functionally identical effects on weight reduction. Here we summarize a universal mechanism in which weight loss is modulated by mitochondrial biogenesis, which is correlated with conversion from the mitochondria-insufficient white adipose tissue to the mitochondria-abundant brown adipose tissue. This mechanistic description of inflammatory obesity may prove useful in the future for guiding pathology-based drug discovery for weight reduction.

Artemisinin: A Panacea Eligible for Unrestrictive Use?

Although artemisinin has been used as anti-malarial drug, accumulating evidence on the extended therapeutic potential of artemisinin emerges. Apart from anti-malaria and anti-tumor, artemisinin can also exert beneficial effects on some metabolic disorders, such as obesity, diabetes, and aging-related diseases. However, whether artemisinin should be applied to treatment of the wide-spectrum diseases is debating. Here, we discuss the predisposition of a raised risk of malarial resistance to artemisinin from consideration of the multi-target and non-specific features of artemisinin.

Akkermansia muciniphila May Determine Chondroitin Sulfate Ameliorating or Aggravating Osteoarthritis.

Chondroitin sulfate (CS) has shown either ameliorating or aggravating effects on osteoarthritis (OA) in separately conducted clinical trials. Because CS is usually administered orally, it should be affected by or would impact on the individual gut microbiota. Evidence is accumulating that CS can nourish sulfatase-secreting bacteria (SSB) and sulfate-reducing bacteria (SRB). To decipher how can an individual gut microbiota determine the clinical values of CS for treatment on OA, we suggest here that CS would give distinct outcomes for OA treatment depending on Akkermansia muciniphila, a gut commensal probiotic bacterial species as optimal presence albeit also behaving as mucus-eroding bacteria (MEB) when abundant presence. Briefly, CS would ameliorate OA if A. muciniphila is present due to without overgrowth of SSB and SRB, whereas CS would aggravate OA if A. muciniphila is absent because of failure in or lack of competition with abundant SSB and SRB. By noting such a frequently ignored phenomenon, we urge the development of non-orally administering CS to minimize its side-effects and extend it to other medicinal applications.

Alzheimer's Disease-like Early-phase Brain Pathogenesis: Self-curing Amelioration of Neurodegeneration from Pro-inflammatory 'Wounding' to Anti-inflammatory 'Healing'.

OBJECTIVE: The etiological initiators of neuroinflammation remain inconclusive, and effective interventions to block neurodegeneration are unavailable. Surprisingly, we found collagen II-combined complete Freund's adjuvant (CC) that usually induces rheumatoid arthritis (RA) also drives Alzheimer's disease (AD)-like neurodegeneration in mice. CC not only upregulates the cerebral pro-inflammatory cytokines including tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8), but also downregulates the cerebral interleukin 10 (IL-10), an anti-inflammatory cytokine, and tyrosine hydroxylase (TH), a ratelimiting enzyme for biosynthesis of the anti-inflammatory neurotransmitter dopamine. In contrast, electroacupuncture (EA) elevates TNF-α/IL-8 and declines IL-10/TH at first, but declines TNF-α/IL-8 and elevates IL-10/TH later. Upon impact on mitochondrial biogenesis, ubiquitination, and autophagy, EA firstly potentates but secondly attenuates CC-triggered signaling cascades leading to oxidation, nitrosylation, hypoxia, and angiogenesis. Eventually, EA compromises neurodegeneration by decreasing amyloid- ß peptide (Aß) and phosphorylated tau protein (p-tau), and also rectifies neuronal dysfunctions by increasing the cholinergic neurotransmitter acetylcholine (Ach) and its rate-limiting biosynthetic enzyme choline acetyltransferase (ChAT). RESULTS: Conclusively, EA initially aggravates and subsequently ameliorates CC-evoked AD-like earlyphase brain pathogenesis via conversion from pro-inflammatory microglia to anti-inflammatory microglia.

[Seasonal Variations of Soil Microbial PLFAs and Soil Enzyme Activity Under the Citrus Plantation in Mt. Jinyun, Chongqing].

The soil microbial community and soil enzyme activity are important parts of soil ecosystem,and they are sensitive to the ecological environment.In this study,citrus plantation in Mt.Jinyun was chosen as the research object.The phospholipid fatty acid (PLFAs) combined with principal component analysis method was used to study the effect of season changes on soil microbial quantity,community structure characteristics and soil enzyme activity at 0-20 cm soil layer under citrus forest.The results obtained from this study were as follows:① The season changes had a significant impact on soil microbes (P<0.05).Moreover,16:0,i17:0,16:1 2OH,18:0,cy19:0ω8c,i17:1ω9c or 16:0 10-methyl were found to be common in all four seasons,and the proportion of each phospholipid fatty acid was 49.57%,41.63%,35.41% and 38.05%,respectively.In various microbial species,the PLFAs content of bacteria was the highest,followed by fungi,and that of actinomycetes was the lowest.Our results revealed that all kinds of micro-organisms showed obvious seasonal variation characteristics.The total PLFAs quantity of citrus forest ranged from 6.868 to 24.085 nmol·g-1,which had a content order of spring >autumn >winter >summer.The PLFAs content of bacteria,G-,G+ and actinomycetes also showed the same variation trend.However,the PLFAs content of fungi showed a different variation trend,i.e.,autumn was the highest,followed by winter and summer,and spring was the lowest.The results suggested that season change also had a significant impact on microbial community functional diversity index.The variation trend of richness index (R) followed the order of spring >winter >autumn >summer.Moreover,the variation trend of diversity index (H') was winter >autumn >spring >summer.Furthermore,the variation trend of evenness index (J) was summer >autumn >winter >spring.The dominance index (D) increased with the change of season.2 Urease showed a change with the seasons following summer >spring >autumn >winter;Soil invertase,soil catalase and acid phosphatase activity changed obviously in autumn,followed by spring,summer,and winter.3 The principal component analysis demonstrated that the PLFAs content of bacteria,G-,G+,actinomycetes and total PLFAs made the greatest contribution to soil fertility followed by that of invertase,catalase,acid phosphatase and fungi.Moreover,our studies showed that the PLFAs content of urease made a minimum contribution to the soil fertility.

[Seasonal Effect of Simulated Nitrogen Deposition on Soil Respiration and Soil Enzyme Activity in Masson Pine Forest in Mt. Jinyun, Chongqing, China].

Soil enzymes involved in the conversion of soil carbon and nitrogen, meanwhile the availability of soil carbon and nitrogen is the base of soil enzymes, yet atmospheric N deposition influences the release of soil CO2 by reduce the activities of soil enzyme. The objective of this study was to investigate the effect of different nitrogen deposition on soil respiration and soil enzymes, and explore the relationship among soil respiration, soil temperature, soil moisture and soil enzymes in the Masson pine forest. The results might provide a reference for further study on the effects of nitrogen deposition on pine forest ecosystem. From May 2014 to July 2015, three nitrogen application treatments and a control treatment were set up: low nitrogen [N5, 20 g·(m2·a)-1], moderate nitrogen [N10, 40 g·(m2·a)-1], high nitrogen [N15, 60 g·(m2·a)-1] and control treatment [N0, 0 g·(m2·a)-1) in the Masson pine forest. We measured soil respiration, soil temperature, and soil moisture simultaneously by using the Automated Soil CO2 Exchange Station (ACE, UK). The results showed that: 1 Soil enzymes and soil respiration had obvious seasonal variation, soil respiration of N0, N5, N10 and N15 was the highest in Summer, followed by the Spring and Autumn, and the lowest in Winter, and no consistent change rule was found in soil enzymes. 2 Generally, nitrogen deposition suppressed soil respiration and soil enzymes, and these inhibitory effects were strengthened with increasing levels of nitrogen deposition. The only exception in which nitrogen deposition enhanced soil respiration was in the Masson pine forest in Winter, In Spring, Summer and Autumn, nitrogen deposition suppressed soil enzymes, while there was difference among Ure, Ive, CAT and ACP in Winter. 3 stepwise regression showed that in control treatment and low nitrogen treatment, T, Ure and Ive made great contributions to the Rs, and Rs rapidly increased with the increase of T, Ure and Ive. In middle nitrogen treatment, T, Ure and CAT made great contributions to the Rs, and Rs increased with the increase of T, Ure and CAT. In high nitrogen treatment, Rs decreased with the increase of Ure, yet Rs increased with the increase of CAT and W.

[Effect of Nitrogen Deposition on Soil Microbial Community Structure Determined with the PLFA Method Under the Masson Pine Forest from Mt. Jinyun, Chongqing].

Nitrogen deposition increases the productivity of forest-land, yet may cause environmental damage, while soil microorganisms are sensitive to the ecological environment. The objective of this study was to investigate the effects of nitrogen deposition on soil microbial communities and the relationship among soil microbial communities, soil temperature, soil moisture and the concentration of nitrogen deposition in the Masson pine forest. The results might provide a reference for further study on the effects of nitrogen deposition on forest ecosystem of Masson pine. From May 2014 to June 2015, three nitrogen application treatments and the control treatment were set up:low nitrogen [N20,20 g·(m2·a)-1], moderate nitrogen [N40, 40 g·(m2·a)-1], high nitrogen [N60, 60 g·(m2·a)-1] and control treatment [N0, 0 g·(m2·a)-1] in the Masson pine forest. Soil microbial community structure, soil temperature and soil moisture were measured by phosphorus lipid fatty acids (PLFAs) and automated soil CO2 exchange station. The results showed that:1 Seasonal variation had a remarkable effect on soil bacteria, fungi, actinomycetes and the total PLFA(P<0.05), all kinds of soil microorganisms had the highest amounts in spring and the lowest in winter. In different seasons, the response of different soil microorganisms to nitrogen deposition was different. Generally, nitrogen deposition decreased soil microorganisms in spring and autumn, while in summer and winter, nitrogen deposition had a positive effect on soil microorganisms. 2 Nitrogen deposition had a significant effect on soil microbial community structure(P<0.05). In spring and summer, low nitrogen and moderate nitrogen decreased the soil microbial richness index and diversity index, yet increased the evenness index. In the autumn and winter, low nitrogen and moderate nitrogen promoted the soil microbial richness index, diversity index and evenness index. In all four seasons, nitrogen deposition reduced the soil microbial richness index, diversity index and evenness index. 3 Correlation analysis showed that the concentration of nitrogen deposition had a very significant negative correlation with soil bacteria (P<0.01), and a significant negative correlation with total PLFA (P<0.05). Soil temperature had a significant negative correlation with soil actinomycetes. Soil moisture had a significant positive correlation with bacteria and total PLFA. In conclusion, soil microorganisms of Masson pine forest were mainly influenced by soil moisture and less affected by soil temperature in Jinyun Mountain. Nitrogen deposition had a significant effect on microbial community structure and diversity.

Artemisinin mimics calorie restriction to trigger mitochondrial biogenesis and compromise telomere shortening in mice.

Calorie restriction is known to extend lifespan among organisms by a debating mechanism underlying nitric oxide-driven mitochondrial biogenesis. We report here that nitric oxide generators including artemisinin, sodium nitroprusside, and L-arginine mimics calorie restriction and resembles hydrogen peroxide to initiate the nitric oxide signaling cascades and elicit the global antioxidative responses in mice. The large quantities of antioxidant enzymes are correlated with the low levels of reactive oxygen species, which allow the down-regulation of tumor suppressors and accessory DNA repair partners, eventually leading to the compromise of telomere shortening. Accompanying with the up-regulation of signal transducers and respiratory chain signatures, mitochondrial biogenesis occurs with the elevation of adenosine triphosphate levels upon exposure of mouse skeletal muscles to the mimetics of calorie restriction. In conclusion, calorie restriction-triggered nitric oxide provides antioxidative protection and alleviates telomere attrition via mitochondrial biogenesis, thereby maintaining chromosomal stability and integrity, which are the hallmarks of longevity.

[Effects of Different Altitudes on Soil Microbial PLFA and Enzyme Activity in Two Kinds of Forests].

The soil microbial community is an important part in soil ecosystem, and it is sensitive to the ecological environment. Phospholipid-derived fatty acids ( PLFA ) analysis was used to examine variations in soil microbial community diversity and its influencing factors. The results showed that: there existed 48 PLFAs that were significant in the soil samples from six altitudes. The PLFAs of six altitudes with the highest contents were i16:0, 10Me17:0, 10Me18:0 TBSA. The citrus forest exhibited richer soil PLFAs distribution both in type and amount than those in masson pine. The microbial activity and functional diversity of masson pine were increased with increasing altitudes, and citrus forest gradually decreased, the PLFA content of different microbial groups in each altitude were significantly different. The richness index, Shannon-Wiener index and Pielou evenness index of masson pine in low elevation were holistically higher than those in high elevation. However, the highest richness index of citrus forest was in low altitude, the highest Shannon-Wiener index and Pielou evenness index were in high altitude. The PLFAs content of different microbial groups were closely correlated to the soil enzyme activities and environmental factors. The PLFAs of bacteria, actinomycetes, G⁻ (Gram- positive), G⁺ (Gram-negative) were positively correlated with Ure(urease) , Ive(invertase) , CAT( catalase activity) and forest type, the PLFAs of fungi was significantly correlated with Ure, Ive, CAT, the PLFAs of bacteria, fungi, actinomycetes, G⁻ , G⁺ were significantly negatively or less correlated with elevation. Ure, Ive, CAT, forest type and elevation are the pivotal factors controlling the soil microbial biomass and activities.

[Influence of different slope position and profile in Disporopsis pernyi forest land on soil microbial biomass and enzyme activity in southwest Karst mountain of China ].

Soil microbial biomass and enzyme activity are important parameters to evaluate the quality of the soil environment. The goal of this study was to determine the influence of different slope position and section in Disporopsis pernyi forest land on the soil microbial biomass and enzyme activity in southwest Karst Mountain. In this study, we chose the Dip forest land at Yunfo village Chengdong town Liangping country Chongqing Province as the study object, to analyze the influence of three different slope positions [Up Slope(US), Middle Slope(MS), Below Slope(BS)] and two different sections-upper layer(0-15 cm) and bottom layer(15-30 cm) on the soil microbial biomass carbon (SMBC), soil microbial biomass nitrogen (SMBN), microbial carbon entropy (qMBC), microbial nitrogen entropy (qMBN) , catalase(CAT), alkaline phosphatase (ALK), urease(URE), and invertase(INV). The results showed that the same trend (BS > MS > US) was found for SMBC, SMBN, qMBC, qMBN, CAT and INV of upper soil layer, while a different trend (BS > US > MS) was observed for ALK. In addition, another trend (MS > US > BS) was observed for URE. The same trend (BS > MS >US) was observed for SMBN, qMBN, CAT, ALK, URE and INV in bottom layer, but a different trend (MS > BS > US) was observed for SMBC and qMBC. The SMBC, SMBN, CAT, ALK, URE and INV manifested as upper > bottom with reduction of the section, while qMBC and qMBN showed the opposite trend. Correlation analysis indicated that there were significant (P <0.05) or highly significant (P < 0.01) positive correlations among SMBC in different slope position and section, soil enzyme activity and moisture. According to the two equations of regression analysis, SMBC tended to increase with the increasing CAT and ALK, while decreased with the increasing pH. Then SMBN tended to increase with the increasing URE and INV.

Nitric oxide-driven hypoxia initiates synovial angiogenesis, hyperplasia and inflammatory lesions in mice.

BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory articular disease with cartilage and bone damage due to hyperplasic synoviocyte invasion and subsequent matrix protease digestion. Although monoclonal antibodies against tumor necrosis factor alpha (TNFα) have been approved for clinical use in patients with RA, desired therapeutic regimens suitable for non-responders are still unavailable because etiological initiators leading to RA remain enigmatic and unidentified. METHODOLOGY/PRINCIPAL FINDINGS: Bacteria-induced arthritis (BIA) that simulates collagen-induced arthritis (CIA) is developed in mice upon daily live bacterial feeding. The morphological lesions of paw erythema and edema together with the histological alterations of synovial hyperplasia and lymphocytic infiltration emerge as the early-phase manifestations of BIA and CIA. Bacteria- or collagen-mediated global upregulation of pro-inflammatory cytokines is accompanied by the burst of nitric oxide (NO). Elevation of the serum NO level is correlated with decline of the blood oxygen saturation percentage (SpO2), reflecting a hypoxic consequence during development towards arthritis. NO-driven hypoxia is further evident from a positive relationship between NO and lactic acid (LA), an end product from glycolysis. Upregulation of hypoxia inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) validates hypoxia-induced angiogenesis in the inflamed synovium of modeling mice. Administration of the NO donor compound sodium nitroprusside (SNP) causes articular inflammation by inducing synovial hypoxia. Anti-bacteria by the antibiotic cefotaxime and/or the immunosuppressant rapamycin or artesunate that also inhibits nitric oxide synthase (NOS) can abrogate NO production, mitigate hypoxia, and considerably ameliorate or even completely abort synovitis, hence highlighting that NO may serve as an initiator of inflammatory arthritis. CONCLUSIONS/SIGNIFICANCE: Like collagen, bacteria also enable synovial lesions via upregulating pro-inflammatory cytokines, triggering NO production, driving hypoxic responses, and inducing synovial angiogenesis and hyperplasia, suggesting that sustained infection might be, in part, responsible for the onset of synovitis and arthritis in mice.

Artesunate potentiates antibiotics by inactivating heme-harbouring bacterial nitric oxide synthase and catalase.

BACKGROUND: A current challenge of coping with bacterial infection is that bacterial pathogens are becoming less susceptible to or more tolerant of commonly used antibiotics. It is urgent to work out a practical solution to combat the multidrug resistant bacterial pathogens. FINDINGS: Oxidative stress-acclimatized bacteria thrive in rifampicin by generating antibiotic-detoxifying nitric oxide (NO), which can be repressed by artesunate or an inhibitor of nitric oxide synthase (NOS). Suppressed bacterial proliferation correlates with mitigated NO production upon the combined treatment of bacteria by artesunate with antibiotics. Detection of the heme-artesunate conjugate and accordingly declined activities of heme-harbouring bacterial NOS and catalase indicates that artesunate renders bacteria susceptible to antibiotics by alkylating the prosthetic heme group of hemo-enzymes. CONCLUSIONS: By compromising NO-mediated protection from antibiotics and triggering harmful hydrogen peroxide burst, artesunate may serve as a promising antibiotic synergist for killing the multidrug resistant pathogenic bacteria.

Artesunate mitigates proliferation of tumor cells by alkylating heme-harboring nitric oxide synthase.

Artesunate (ART), a semi-synthetic derivative of antimalarial artemisinin, kills cancer cells with uncertain mechanisms. Here, we report for the first time that ART may exert the anti-tumor activity by conjugating the prosthetic heme of hemoproteins in a hepatoma cell line, HepG2, which was evident by monitoring the shift of absorbance from heme (A415) to the ART-heme adduct (A476). Accordingly, a transient elevation of A415 was observed with a synchronous burst of nitric oxide (NO) and a high rate of survival following incubation of HepG2 with 50 µM ART. In contrast, ART at above 100 µM led to an abrogation of NO generation and a decline of the survival rate in HepG2. These data implied that heme-containing nitric oxide synthase (NOS) may represent a major cellular target of ART in killing tumor cells.

Senescent leaves of Artemisia annua are one of the most active organs for overexpression of artemisinin biosynthesis responsible genes upon burst of singlet oxygen.

To dissect and penetrate complexicity regarding the tissue-specific and environment-induced expression modes of cytosolic and plastidial terpene biosynthetic genes in A. annua, corresponding mRNAs relevant to terpene biosynthesis were quantitatively compared among distinctive organs and during different growth stages. Although all examined mRNAs gradually elevate from June to August in tested organs, a putative artemisinin biosynthesis responsible DBR2 mRNA represents the most abundant transcript anyplace and anytime. Apart from others, senescent leaves endow global activation of artemisinin biosynthetic genes and ultimately lead to enhanced artemisinin production. Direct measurement of (1)O (2) burst from senescent leaves strongly supports an involvement of (1)O (2) in conversion from precursor(s) to artemisinin. In the context of environmental stresses, physical and chemical stress signals that include those invoking (1)O (2) burst were evaluated as if inducing artemisinin biosynthetic genes. The quantitative data have reiterated a common pattern of modulating artemisinin production in A. annua by triggering (1)O (2) burst during senescence and under chilling acclimatization. In conclusion, a missing link concatenating senescence-coupled (1)O (2) generation to (1)O (2)-induced upregulation of artemisinin biosynthetic genes has been re-established, which would provide a fertile base for future endeavors pursuing further enhancements of artemisinin production.

Quantitative transcript profiling reveals down-regulation of A sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants.

To investigate the dynamic fluctuation of terpenoid relevant transcriptomics in transgenic ARTEMISIA ANNUA plants that express the genomic integrated antisense squalene synthase gene ( ASSS), we have quantified the transcript levels of the sterol anabolic SS gene as well as artemisinin biogenetic amorphadiene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 reductase (CPR) genes by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). The SS mRNA level in transgenic plants sharply droped to 7.4 % - 55.3 % (i. e., 44.7 - 92.6 % reduction as the wild-type control), strongly implying that the expression of endogenous SS gene is significantly suppressed by the exogenous ASSS gene. In a synchronous fashion, ADS, CYP71AV1 and CPR mRNA levels elevated with the decline of SS mRNA level in transgenic plants, and the maximal ADS, CYP71AV1 and CPR mRNA levels in transgenic plants were 3.0-, 4.4- and 2.5-fold, respectively, higher than those in the control. Without a lethal effect but with a distinguishable impact on the organogenesis and morphology of transgenic plants, the down-regulation of SS gene has also led to the coordinated overexpression of ADS, CYP71AV1 and CPR genes together with the overproduction of artemisinin although no fully perfect correlation among the available experimental data has been shown.

[Cloning, soluble expression and mutant activity analysis of lactate dehydrogenase gene from Plasmodium falciparum].

To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors towards anti-malarial drugs, a lactate dehydrogenase gene of Plasmodium falciparum (PfLDH) was amplified from the Hainan isolate FCC1/HN. The fusion expression vectors, pGEX-2TK and pET-29a( + ), were utilized to introduce the PfLDH gene into strains of Escherichia coli, BL21 and BL21 (DE3), for over-expression. Consequently, the enzymatic activity of PfLDH was successfully detected in the suspension of lytic bacteria. The PfLDH gene cloned in pGEX-2TK was mainly expressed as inclusion bodies, while the same gene cloned in pET-29a( + ) was nearly expressed in a soluble form of PfLDH, demonstrating the latter vehicle might be more suitable for the large-scale preparation of recombinant PfLDH. Furthermore, according to the electrophoregram of SDS-PAGE and the sequencing data, a series of truncated PfLDH sequences generated randomly from gene amplification were screened and cloned, from which four pre-matured genes with a terminator mutation, PfLDH-delta271, -delta236, -delta167 and -delta53 coding for 45, 80, 149 and 263 amino acid residues, were individually recovered. Through the gene expression and enzymatic activity measurement, the effect of pre-matured terminator mutation on the activity of PfLDH was evaluated, which should pave the way for probing the relationship between structure and function of PfLDH.