[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

[Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro].

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Cognition contra camouflage: How the brain mediates predator-driven crypsis evolution.

While crypsis is a prominent antipredator adaptation, the role of the brain in predator-driven evolution remains controversial. Resolving this controversy requires contextualizing the brain with established antipredator traits and predation pressure. We hypothesize that the reduced predation risk through crypsis relaxes predation-driven selection on the brain and provide comparative evidence across 102 Chinese frog species for our hypothesis. Specifically, our phylogenetic path analysis reveals an indirect relationship between predation risk and crypsis that is mediated by brain size. This result suggests that at a low predation risk, frogs can afford to be conspicuous and use their large brain for cognitive predator evasion. This strategy may become less efficient or energetically costlier under higher predation pressure, favoring smaller brains and instead increasing crypsis.

Global view on virus infection in non-human primates and implications for public health and wildlife conservation.

Viruses can be transmitted from animals to humans (and vice versa) and across animal species. As such, host-virus interactions and transmission have attracted considerable attention. Non-human primates (NHPs), our closest evolutionary relatives, are susceptible to human viruses and certain pathogens are known to circulate between humans and NHPs. Here, we generated global statistics on VI-NHPs based on a literature search and public data mining. In total, 140 NHP species from 12 families are reported to be infected by 186 DNA and RNA virus species, 68.8% of which are also found in humans, indicating high potential for crossing species boundaries. The top 10 NHP species with high centrality in the NHP-virus network include two great apes (Pan troglodytes, Pongo pygmaeus) and eight Old World monkeys (Macaca mulatta, M. fascicularis, M. leonina, Papio cynocephalus, Cercopithecus ascanius, C. erythrotis, Chlorocebus aethiops, and Allochrocebus lhoesti). Given the wide distribution of Old World monkeys and their frequent contact with humans, there is a high risk of virus circulation between humans and such species. Thus, we suggest recurring epidemiological surveillance of NHPs, specifically Old World monkeys that are in frequent contact with humans, and other effective measures to prevent potential circulation and transmission of viruses. Avoidance of false positives and sampling bias should also be a focus in future work.

Proximate causes of dispersal for female Yunnan snub-nosed monkeys.

Individual dispersal trends, unquestionably important for species ecology and evolution, are affected by multiple factors. Understanding the factors that influence female dispersal strategies offers important insight into primate dispersal mechanisms and female choice. To investigate the proximate causes of dispersal in female Yunnan snub-nosed monkeys (Rhinopithecus bieti), we observed and analyzed nine years of detailed dispersal and demographic data from a population of R. bieti in Xiangguqing, Baimaxueshan Nature Reserve, Yunnan Province, China. Results showed that females who lived long-term in a one-male unit (OMU), without giving birth and with few or no relatives, were more likely to leave that OMU. In addition, an OMU led by an outgroup male and containing more female relatives was significantly more likely to be chosen for immigration. Conversely, greater male age, longer male tenure, and more potentially fertile females discouraged immigration into an OMU. These results suggest that reproduction, male quality, and kin cooperation play the largest roles in female Yunnan snub-nosed monkey dispersal.

Chronic ethanol exposure facilitates facial-evoked MLI-PC synaptic transmission via nitric oxide signaling pathway in vivo in mice.

Ethanol (EtOH) exposure causes alterations of motor coordination, balance, behavior, speech, and certain cognitive functions are considered to be caused partly by impairment of cerebellar circuits function and modulation of synaptic transmission. The cerebellar cortical molecular layer interneuron-Purkinje cell (MLI-PC) synapses are critical for various information integration and transmission, which are sensitive to acute and chronic EtOH exposure. The aim of this study is to investigate the effect of chronic ethanol exposure on the facial stimulation-evoked MLI-PC synaptic transmission in urethane-anesthetized mice, by electrophysiological recording and pharmacological methods. Under current-clamp recording conditions, air-puff stimulation of ipsilateral whisker pad evoked MLI-PC synaptic transmission, which expressed an inhibitory component (P1) followed by a pause of simple spike (SS) firing in cerebellar PCs. Chronic ethanol exposure did not change the latency of the facial stimulation-evoked responses in cerebellar PCs, but induced significant enhancement of the stimulation-evoked MLI-PC synaptic transmission, which expressed increases in amplitude of P1 and pause of SS firing. The amplitude of P1 and pause of SS in ethanol exposure group were significant higher than that in control group. Cerebellar surface application of nitric oxide synthesis (NOS) inhibitor, L-NNA (5 mM) significantly decreased the amplitude of P1 and the pause of SS firing in EtOH exposure group, but did no effect on control group. In contrast, cerebellar surface application of NO donor, SNAP (100 µM) significantly increased the amplitude of P1 and the pause of SS firing in control group, but not in EtOH exposure group. These results indicated that chronic EtOH exposure significantly facilitated the sensory-evoked MLI-PC synaptic transmission via NO signaling pathway in mouse cerebellar cortex.

Preliminary report on Sichuan golden snub-nosed monkeys (Rhinopithecus roxellana roxellana) at Laohegou Nature Reserve, Sichuan, China.

Comparative studies of subspecies under different ecological environments offer insights into intraspecies evolutionary adaptive mechanisms. Golden snub-nosed monkeys (Rhinopithecus roxellana) include three subspecies in China classified mainly by their morphological variations: R. r. roxellana (Sichuan and Gansu province), R. r. qinlingensis (Shaanxi province) and R. r. hubeiensis (Hubei province). These three subspecies live in three isolated area with different environments. Past works focused on the last two subspecies, but little information of habitat and behaviors of the nominated subspecies (R. r. roxellana) is available to date. We conducted a two-year study on the diet, activity budget, home range and social organization of 4 herds of R. r. roxellana, based on a total of 106 days' observation in Laohegou (LHG) Nature Reserve, Sichuan province. By using scan sampling method, our results suggest that the R. r roxellana feeds predominantly on leaves (77.5%), and spends more time feeding (40.0%) and resting (27.0%) while compared to the other two subspecies. Kernel Density Estimation Method based on GPS technology confirms that R. r roxellana has relatively larger home ranges (49.1 km2). The unit size (8.3 ± 3.5 individuals) of R. r roxellana is also smaller. Therefore, it is possible that differences in food availability in relation to habitats have important impacts on the feeding strategy and social system of the golden snub-nosed monkey. These results provide data to further explore intraspecific adaptations of living primates.

Large-brained frogs mature later and live longer.

Brain sizes vary substantially across vertebrate taxa, yet, the evolution of brain size appears tightly linked to the evolution of life histories. For example, larger brained species generally live longer than smaller brained species. A larger brain requires more time to grow and develop at a cost of exceeded gestation period and delayed weaning age. The cost of slower development may be compensated by better homeostasis control and increased cognitive abilities, both of which should increase survival probabilities and hence life span. To date, this relationship between life span and brain size seems well established in homoeothermic animals, especially in mammals. Whether this pattern occurs also in other clades of vertebrates remains enigmatic. Here, we undertake the first comparative test of the relationship between life span and brain size in an ectothermic vertebrate group, the anuran amphibians. After controlling for the effects of shared ancestry and body size, we find a positive correlation between brain size, age at sexual maturation, and life span across 40 species of frogs. Moreover, we also find that the ventral brain regions, including the olfactory bulbs, are larger in long-lived species. Our results indicate that the relationship between life history and brain evolution follows a general pattern across vertebrate clades.

Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice.

The cerebellum is sensitive to ethanol (EtOH) consumption. Chronic EtOH consumption impairs motor learning by modulating the cerebellar circuitry synaptic transmission and long-term plasticity. Under in vitro conditions, acute EtOH inhibits both parallel fiber (PF) and climbing fiber (CF) long-term depression (LTD). However, thus far it has not been investigated how chronic EtOH consumption affects sensory stimulation-evoked LTD at the molecular layer interneurons (MLIs) to the Purkinje cell (PC) synapses (MLI-PC LTD) in the cerebellar cortex of living animals. In this study, we investigated the effect of chronic EtOH consumption on facial stimulation-evoked MLI-PC LTD, using an electrophysiological technique as well as pharmacological methods, in urethane-anesthetized mice. Our results showed that facial stimulation induced MLI-PC LTD in the control mice, but it could not be induced in mice with chronic EtOH consumption (0.8 g/kg; 28 days). Blocking the cannabinoid type 1 (CB1) receptor activity with AM-251, prevented MLI-PC LTD in the control mice, but revealed a nitric oxide (NO)-dependent long-term potentiation (LTP) of MLI-PC synaptic transmission (MLI-PC LTP) in the EtOH consumption mice. Notably, with the application of a NO donor, S-nitroso-N-Acetyl-D, L-penicillamine (SNAP) alone prevented the induction of MLI-PC LTD, but a mixture of SNAP and AM-251 revealed an MLI-PC LTP in control mice. In contrast, inhibiting NO synthase (NOS) revealed the facial stimulation-induced MLI-PC LTD in EtOH consumption mice. These results indicate that long-term EtOH consumption can impair the sensory stimulation-induced MLI-PC LTD via the activation of a NO signaling pathway in the cerebellar cortex in vivo in mice. Our results suggest that the chronic EtOH exposure causes a deficit in the cerebellar motor learning function and may be involved in the impaired MLI-PC GABAergic synaptic plasticity.

Effects of continuous positive airway pressure therapy on plasma aldosterone levels in patients with obstructive sleep apnea: A meta-analysis.

Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular diseases. Aldosterone was reported to be increased in patients with OSA and correlated with OSA severity. Many studies investigated the effect of continuous positive airway pressure (CPAP) therapy on plasma aldosterone concentrations (PAC) in OSA patients. The results, however, were inconsistent. In the present study, we aimed to evaluate the effects of CPAP therapy on PAC by performing a meta-analysis. Literature search was carried out in electronic databases including PubMed/Medline, Cochrane Library, Embase and Web of Science. Eligible full-text articles were identified, and important data were extracted. Pooled analysis was performed using the STATA12.0 and RevMan 5.2. Standardized mean difference (SMD) was calculated to estimate the treatment effects. A total of eight studies involving 219 patients were included for our final analysis. PAC was found unchanged after CPAP treatment in OSA patients (SMD=-0.36, 95% CI:-0.91 to 0.18, Z=1.32, P=0.19). Meanwhile, CPAP therapy showed no impact on PAC (SMD=-0.21, 95% CI:-0.85 to 0.42, Z=0.66, P=0.51) in a separate meta-analysis including 3 randomized controlled trials. In conclusion, the evidence for the use of CPAP therapy to decrease PAC in OSA patients is low, and further studies are still warranted.

[Correlation between autophagy and polarization of macrophages in atherosclerosis plaque in arteriosclerosis obliterans amputees].

This study was designed to investigate the correlation between autophagy and polarization of macrophages in atherosclerosis (AS) plaque in arteriosclerosis obliterans amputees. Femoral artery specimens from arteriosclerosis obliterans amputees were performed hematoxylin and eosin (HE) staining, oil red O and immunofluorescence staining to observe the morphology of atherosclerotic plaque, phenotype of macrophages and autophagy in plaque; using real-time quantitative RT-PCR technology to detect the mRNA level of M1 and M2 type markers in arterial tissue; to analyze polarized signal pathway and autophagy protein levels in macrophages by Western blotting. Arterial specimens staining showed obvious lipid deposition and obvious infiltration of amount of foam cells and inflammatory cells. Macrophages were mainly expression M1 type in percentage in fibrous plaque. Although both M1 and M2 macrophages were upregulated in atheromatous plaque, the increase was dominant in M2 type in percentage. The level of autophagy was significantly higher in the atheromatous plaque than that of fibrous plaque. The expression of tumor necrosis factor- α (TNF-α), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA was significantly higher in fibrous plaque than that of atheromatous plaque (P < 0.01 or 0.05), and arginase-1 (Arg-1), transforming growth factor-ß (TGF-ß), CD163 and interleukin-10 (IL-10) mRNA was significantly lower than that in atheromatous plaque (P < 0.01). The levels of p-STAT1 and NF-κB were significantly increased in fibrous plaque (P < 0.01), while p-STAT6 expression was significantly increased in atheromatous plaque (P < 0.01). The level of LC3-II was significantly higher in atheromatous plaque than that in fibrous plaque (P < 0.01). Macrophages in early atherosclerotic plaque were induced to M1 type through p-STAT1/NF-κB pathway and expressed moderate levels of autophagy; while macrophages in advanced plaques were induced to polarization of M2 type through p-STAT6 pathway. M2 macrophages expressed a higher level of autophagy than M1 macrophages.

A new method for evaluating regional cerebral blood flow changes: Laser speckle contrast imaging in a C57BL/6J mouse model of photothrombotic ischemia.

The present study aimed to improve the processing of data acquired from laser speckle contrast imaging (LSCI) to provide a standardization method to explore changes in regional cerebral blood flow (rCBF) and to determine the correlations among rCBF, cerebral ischemic lesion volume and microvascular density over time in a focal ischemic region. C57BL/6J mice were subjected to focal photothrombotic (PT) ischemia. rCBF was measured using LSCI at different time points before and after PT ischemia through an intact skull. Standardized rCBF (SrCBF), defined as the ratio of rCBF measured in the ipsilateral region of interest (ROI) to that in the corresponding contralateral region, was calculated to evaluate potential changes. In addition, the volume of the ischemic lesion and the microvascular density were determined using Nissl staining and immunofluorescence, respectively. The relationships among the ischemic lesion volume, microvascular density and SrCBF were analyzed over time. The results showed that the cortical rCBF measured using LSCI following PT ischemia in the C57BL/6J mice gradually increased. Changes in the cerebral ischemic lesion volume were negatively correlated with SrCBF in the ischemic region. Changes in the microvascular density were similar to those observed in SrCBF. Our findings indicate that LSCI is a practical technique for observing changes in murine cortical rCBF without skull opening and for analyzing the relationships among the ischemic lesion volume, microvascular density and SrCBF following focal cerebral ischemia. Preliminary results also suggest that the use of LSCI to observe the formation of collateral circulation is feasible.

[Effect of ASO Blood Stasis Syndrome Serum on Vascular Endothelial Cell Injury and Regulation of Taohong Siwu Decoction on it].

OBJECTIVE: To explore the effect of arteriosclerosis obliterans (ASO) blood stasis syndrome (BSS) serum on vascular endothelial cell injury and to study the regulation of Taohong Siwu Decoction (TSD) on it. METHODS: Umbilical vein endothelial cell culture system was established. The serum endothelial cell injury model with ASO BSS was prepared. Low, medium, and high concentrations TSD containing serums were respectively added. The endothelial cell proliferation activity was observed by MTT method. Ultrastructures of endothelial cells were observed under transmission electron microscope. Changes of intracellular calcium ion concentration and the cytoskeleton were observed under laser confocal microscope. Contents of ET, NO, and transforming growth factor beta1 (TGF-beta1) in endothelial cell culture supernatant were detected by ELISA. RESULTS: In ASO BSS serum group endothelial cell proliferation activities decreased, the cell structure was obviously destroyed, calcium ion concentration increased, contents of ET, NO and TGF-beta1 increased significantly (P < 0.01), and ET/NO ratio was imbalanced. After incubating with TSD drug containing serum, endothelial cell proliferation activities and injured cell structures were obviously improved; ET, NO and TGF-beta1 levels decreased (P < 0.05, P < 0.01), ET/NO ratios approximated to the normal level. CONCLUSION: The main mechanism of TSD for treating ASO ASS lied in improving injured vascular endothelial cells and endocrine disorder.

Tomato WRKY transcriptional factor SlDRW1 is required for disease resistance against Botrytis cinerea and tolerance to oxidative stress.

WRKY proteins comprise a large family of transcription factors that play important roles in plant responses to biotic and abiotic stresses; however, only a few of tomato WRKYs have been studied for their biological functions. In the present study, we identified a Botrytis cinerea-responsive WRKY gene SlDRW1 (Solanum lycopersicumdefense-related WRKY1) from tomato. SlDRW1 is a nucleus localized protein with transactivation activity in yeast. Expression of SlDRW1 was significantly induced by B. cinerea, leading to 10-13 folds of increase than that in the mock-inoculated plants but not by Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlDRW1 resulted in increased severity of disease caused by B. cinerea, but did not affect the phenotype of disease caused by Pst DC3000. In addition, silencing of SlDRW1 also resulted in decreased tolerance against oxidative stress but did not affect drought stress tolerance. Furthermore, silencing of SlDRW1 attenuated defense response such as expression of defense-related genes after infection by B. cinerea. Our results demonstrate that SlDRW1 is a positive regulator of defense response in tomato against B. cinerea and oxidative stress.

[Taohong Siwu Decoction regulated functions of endothelial cells and treated arteriosclerosis obliterans: an experimental study].

OBJECTIVE: To discuss the effect of Taohong Siwu Decoction (TSD) in regulating functions of endothelial cells and treating arteriosclerosis obliterans (ASO). METHODS: The ASO model was prepared by using high-fat diet plus intimal injury. They were randomly divided into the model group (n = 10), the normal control group (n = 9), the low dose TSD group (group A, n = 12), the middle dose TSD group (group B, n = 10), and the high dose TSD group (group C, n = 9). Eight weeks after modeling, the limb blood perfusion was observed using laser Doppler flowmetry. The arterial morphology was observed using light microscope and transmission electron microscope. The number of circulating endothelial cells (CECs) was determined using Percoll density gradient centrifugation method. Serum levels of TNF-alpha, IL-1, ET-1, and NO were detected using double antibody sandwich assay of enzyme linked immunosorbent assay (ELISA). RESULTS: The ASO rat model was successfully established. Blood lipids levels significantly increased, the blood perfusion of left hind limbs significantly decreased, the number of CECs in the peripheral blood significantly increased, the arterial lumen was irregularly narrowed, the ultra-structure of vessel walls was damaged, serum levels of TNF-alpha, IL-1, and ET-1 significantly increased, and the serum level of NO significantly decreased in the model group, showing statistical difference when compared with the normal control group (P < 0.01). Compared with the model group, significant improvement in the aforesaid indices was shown in group B and C (P < 0.05, P < 0.01). CONCLUSIONS: The injury and abnormal functions of endothelial cells is an important pathological process of ASO. As an effective recipe for treating ASO, TSD could protect vascular endothelial cells and improve the secretion function of vascular endothelial cells.

[Functions of NAC transcription factors in biotic and abiotic stress responses in plants].

NAC transcription factors belong to a unique class of transcription factors in plants. The common characteristics of the NAC proteins are the presence of a conserved NAC domain, comprising of about 150 amino acids in N-terminals and a highly variable transcriptional regulation region in C-terminals. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. In this minireview, we summarized the functions and mechanisms of the NAC transcriptional factors in plant abiotic and biotic stress responses. We also discussed future directions towards understanding the biological functions of the members of the NAC transcriptional factors in plants.

Niche partitioning between sympatric rhesus macaques and Yunnan snub-nosed monkeys at Baimaxueshan Nature Reserve, China.

Here we provide a preliminary assessment of dietary and habitat requirements of two sympatric primate taxa, a "simple-stomached" and "complex-stomached" species (Rhinopithecus bieti Colobinae vs. Macaca mulatta Cercopithecinae), as a basis for illuminating how the two coexist. Of ca. 22 plant food species consumed by the macaques, at least 16 were also eaten by the snub-nosed monkeys. Both species showed a preference for fruits. While the snub-nosed monkeys did not utilize any resources associated with human communities, rhesus macaques did occasionally raid agricultural crops. The mean elevation of the snub-nosed monkey group was 3,218 m, while the mean elevation of the macaque group was 2,995 m. Macaques were also spotted on meadows whereas snub-nosed monkeys evidently avoided these. For both species, mixed deciduous broadleaf/conifer forest was the most frequently used ecotype, but whereas evergreen broadleaf forest (Cyclobalanopsis community) accounted for only 3% of the location records of the snub-nosed monkeys, it accounted for 36% of the location records of the macaques. Groups of the two species usually kept a considerable spatial distance from one another (mean 2.4 km). One close encounter and confrontation between groups of the two species resulted in the macaque group moving away. Our findings suggest that the coexistence of the two taxa is facilitated via differential macrohabitat use and spatial avoidance. Although divergent habitat-use strategies may reflect interspecific competition, they may also merely reflect different physiological or ecological requirements.

[Effect of gene transfer using nanoparticles as gene vector in different animal models].

OBJECTIVE: To evaluate the effect of antisense monocyte chemotactic protein-1 (A-MCP-1) nanoparticles (NPs) as gene carrier on gene transfer in two kinds of animal models. METHODS: Poly (lactic acid-co-glycolic acid) (PLGA) was used to make the NPs loaded with A-MCP-1 through a double-emulsion/solvent evaporation technique. NPs size was assessed by dynamic laser defractometer. The particle morphology was observed by scanning electron microscopy. DNA content in the NPs was measured by dissolving known amounts of NPs in chloroform and extracting DNA with water. In vitro release was performed in tris-EDTA buffer at 37 degrees C using double-chamber diffusion cells. The receiver buffer was replaced daily. The A-MCP-1 NPs was transfected into the cultured smooth muscle cells. PCR was used to evaluate the transfection of A-MCP-1. Cationic lipid (Lipofectamine) was used to transfect A-MCP-1 as control. After 48 hours incubation, cells were digested and examined by polymerase chain reaction. Twenty New Zealand white rabbits under jugular vein to artery bypass grafting procedure were divided into four groups: the first group received grafts treated with A-MCP-1 NPs, the second group received grafts treated with cationic liposome (dioleoyl trimethyl ammonium propane)-A-MCP-1, the third group received grafts treated with plasmid DNA, and the fourth group received grafts without transfection as control. Fourteen days after surgery the grafts were harvested. The expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blotting. The morphology of the grafts was investigated. To establish abdominal aortic aneurysms rats model, rats were randomly divided into three groups: A-MCP-1 NPs injection group, shame NPs injection group and control groups (without injection). Two weeks after surgery, diameter of abdominal aorta was measured and aortic tissue was obtained for PCR analysis to evaluate the A-MCP-1 expression. Western blot were applied to detect the inhibitory effect to the expression of MCP-1 mRNA and CD68 protein by A-MCP-1 NPs. RESULTS: NPs size ranged 198nm to 205nm with average around 201.4 nm. DNA content in the NPs was 4.14%. NPs showed steady release rate in vitro in Tris-EDTA solution. It released faster in the first week then maintained a slowly sustained release up to 16 days. In cell culture A-MCP-1 gene successfully transfected into smooth muscle cells by NPs vector. In vein grafting animal model, A-MCP-1 expression was detected in the vascular walls of NPs and cationic lipid treated groups. The degree of vascular hyperplasia in the gene NPs treated group was significantly lower than that in control group. There was no significant difference in the inhibition of intimal hyperplasia between NPs and cationic lipid treated groups. Two weeks after transfection in abdominal aortic aneurysm rats models, the abdominal aortic diameter of A-MCP-1 NPs injection group was (1.79 +/- 0.12) mm, significantly smaller than that of control groups [shame NPs group was (2.58 +/- 0.21) mm, and saline group was (2.63 +/- 0.29) mm] (P < 0.01). The expressions of MCP-1 mRNA and CD68 protein in A-MCP-1 NPs injection group were 12.5 +/- 1.5 and 17.6 +/- 2.1, which were much lower than those in control group [in shame NPs group, which were 35.7 +/- 4.5, 42.3 +/- 5.7 (P < 0.01), and saline group which is 32.4 +/- 3.9, 39.8 +/- 4.8 (P < 0.01)]. Specific band of A-MCP-1 was detected only in the A-MCP-1 NPs injection group by PCR. CONCLUSION: A-MCP-1 gene NPs can be successfully used in rabbit vein grafting model and abdominal aortic aneurysm rats models, and may be potentially applied in clinical practice.

Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAgamma1.

The recessive gene xa5 for resistance to bacterial blight resistance of rice is located on chromosome 5, and evidence based on genetic recombination has been shown to encode a small subunit of the basal transcription factor IIA (Iyer and McCouch in MPMI 17(12):1348-1354, 2004). However, xa5 has not been demonstrated by a complementation test. In this study, we introduced the dominant allele Xa5 into a homozygous xa5-line, which was developed from a cross between IRBB5 (an indica variety with xa5) and Nipponbare (a japonica variety with Xa5). Transformation of Xa5 and subsequent segregation analysis confirmed that xa5 is a V39E substitution variant of the gene for TFIIAgamma on chromosome 5 (TFIIAgamma5 or Xa5). The rice has an addition gene for TFIIAgamma exists on chromosome 1 (TFIIAgamma1). Analysis of the expression patterns of Xa5 (TFIIAgamma5)/xa5 and TFIIAgamma1 revealed that both the genes are constitutively expressed in different rice organs. However, no expression of TFIIAgamma1 could be detected in the panicle by reverse transcriptase-polymerase chain reaction. To compare the structural difference between the Xa5/xa5 and TFIIAgamma1 proteins, 3-D structures were predicted using computer-aided modeling techniques. The modeled structures of Xa5 (xa5) and TFIIAgamma1 fit well with the structure of TFIIA small subunit from human, suggesting that they may all act as a small subunit of TFIIA. The E39V substitution in the xa5 protein occurs in the alpha-helix domain, a supposed conservative substitutable site, which should not affect the basal transcription function of TFIIAgamma. The structural analysis indicates that xa5 and Xa5 potentially retain their basic transcription factor function, which, in turn, may mediate the novel pathway for bacterial blight resistance and susceptibility, respectively.