葛根素对肥胖型高血压大鼠血压和血管功能影响的实验研究

目的:本研究旨在观察饮食中添加葛根素对肥胖型高血压大鼠的心血管代谢指标的影响,尤其关注其对于血压和血管功能的效应。方法:①自发性高血压大鼠24只,分正常饮食对照组(8只)、高脂饮食组(8只)、高脂饮食+葛根素组(8只),大鼠先进行1周的适应性喂养,1周后进行干预,干预时间为14周;②每周测1次体重、鼠尾血压;③实验结束时空腹取血浆测血脂、血糖值,取胸主动脉观察主动脉的内皮依赖性及非内皮依赖性舒张功能。结果:①葛根素可防止高脂饮食导致的自发性高血压大鼠体重的增加及血压、血糖的升高,与高脂饮食组比较,P<0.05或P<0.01。②长期葛根素喂养可有效防止高脂饮食导致的高血压大鼠的血脂水平升高;③长期的葛根素喂养可显著改善肥胖型高血压大鼠的血管舒张功能及降低血压。结论:葛根素可有效改善肥胖型高血压大鼠的相关代谢指标,并可明显降低血压及改善血管功能,提示葛根素对肥胖型高血压有较好的防治作用,值得进一步深入研究。     

miR-375在AGEs介导糖尿病血管损伤细胞中的生物学功能

目的：探讨miR-375在血管损伤细胞中的表达及生物学功能。方法：利用基因克隆技术构建miR-375表达载体;然后将miR-375表达质粒转染至血管损伤细胞中,同时分别设立Huvec12对照组,血管损伤细胞组,血管损伤抑制组,Huvec12转染miR-375组。24h后收集细胞,在mRNA和蛋白水平检测Mtpn、NFκB、profilin1、sICAM1的表达,经荧光染色观察细胞F-actin的变化,再用流式细胞仪检测细胞凋亡。结果：血管损伤细胞中过表达miR-375后,在mRNA和蛋白水平靶基因Mtpn下降,NFκB的表达活性下降,使糖尿病血管病变的标志profilin1下调;F-actin表达恢复;细胞粘附因子（sICAM1）表达下降,细胞凋亡减少。结论：初步证明miR-375可以抑制AGEs介导的糖尿病血管细胞损伤的发生,可能成为糖尿病血管损伤并发症基因治疗的靶点。     

The Chronic Effects of Whey Proteins on Blood Pressure,Vascular Function,and Inflammatory Markers in Overweight Individuals

Limited evidence suggests that dairy whey protein may be the major dairy component that is responsible for health benefits currently associated with increased dairy consumption. Whey proteins may reduce blood pressure and improve cardiovascular health. This study evaluated the effects of whey protein supplementation on blood pressure, vascular function and inflammatory markers compared to casein and glucose (control) supplementation in overweight/obese individuals. The subjects were randomized to either whey protein, casein or glucose supplementation for 12 weeks according to a parallel design. In all, 70 men and women with a mean (±s.e.m.) BMI (kg/m²) of 31.3 ± 0.8 completed the study. Systolic blood pressure (SBP) decreased significantly at week 6 compared to baseline in the whey and casein groups, (P = 0.028 and P = 0.020, respectively) and at week 12 (P = 0.020, and P = 0.017, respectively). Diastolic blood pressure (DBP) decreased significantly compared to baseline in the whey and casein groups (P = 0.038 and P = 0.042, respectively) at week 12. DBP decreased significantly in the whey and casein groups (P = 0.025, P = 0.038, respectively) at week 12 compared to the control group. Augmentation index (AI) was significantly lower from baseline at 12 weeks (P = 0.021) in the whey group. AI decreased significantly in the whey group at 12 weeks compared to control (P = 0.006) and casein (P = 0.006). There were no significant changes in inflammatory markers within or between groups. This study demonstrated that supplementation with whey protein improves blood pressure and vascular function in overweight and obese individuals.     

Blood Pressure-Lowering Peptides from Neo-Fermented Buckwheat Sprouts: A New Approach to Estimating ACE-Inhibitory Activity

Neo-fermented buckwheat sprouts (neo-FBS) contain angiotensin-converting enzyme (ACE) inhibitors and vasodilators with blood pressure-lowering (BPL) properties in spontaneously hypertensive rats (SHRs). In this study, we investigated antihypertensive mechanisms of six BPL peptides isolated from neo-FBS (FBPs) by a vasorelaxation assay and conventional in vitro, in vivo, and a new ex vivo ACE inhibitory assays. Some FBPs demonstrated moderate endothelium-dependent vasorelaxation in SHR thoracic aorta and all FBPs mildly inhibited ACE in vitro. Orally administered FBPs strongly inhibited ACE in SHR tissues. To investigate detailed ACE-inhibitory mechanism of FBPs in living body tissues, we performed the ex vivo assay by using endothelium-denuded thoracic aorta rings isolated from SHRs, which demonstrated that FBPs at low concentration effectively inhibited ACE in thoracic aorta tissue and suppressed angiotensin II-mediated vasoconstriction directly associated with BPL. These results indicate that the main BPL mechanism of FBP was ACE inhibition in living body tissues, suggesting that high FBP''s bioavailability including absorption, tissue affinity, and tissue accumulation was responsible for the superior ACE inhibition in vivo. We propose that our ex vivo assay is an efficient and reliable method for evaluating ACE-inhibitory mechanism responsible for BPL activity in vivo.     

Neuroendocrine Humoral and Vascular Components in the Pressor Pathway for Brain Angiotensin II: A New Axis in Long Term Blood Pressure Control

Central nervous system (CNS) administration of angiotensin II (Ang II) raises blood pressure (BP). The rise in BP reflects increased sympathetic outflow and a slower neuromodulatory pressor mechanism mediated by CNS mineralocorticoid receptors (MR). We investigated the hypothesis that the sustained phase of hypertension is associated also with elevated circulating levels of endogenous ouabain (EO), and chronic stimulation of arterial calcium transport proteins including the sodium-calcium exchanger (NCX1), the type 6 canonical transient receptor potential protein (TRPC6), and the sarcoplasmic reticulum calcium ATPase (SERCA2). Wistar rats received a chronic intra-cerebroventricular infusion of vehicle (C) or Ang II (A, 2.5 ng/min, for 14 days) alone or combined with the MR blocker, eplerenone (A+E, 5 µg/day), or the aldosterone synthase inhibitor, FAD286 (A+F, 25 µg/day). Conscious mean BP increased (P<0.05) in A (123±4 mm Hg) vs all other groups. Blood, pituitary and adrenal samples were taken for EO radioimmunoassay (RIA), and aortas for NCX1, TRPC6 and SERCA2 immunoblotting. Central infusion of Ang II raised plasma EO (0.58±0.08 vs C 0.34±0.07 nM (P<0.05), but not in A + E and A + F groups as confirmed by off-line liquid chromatography (LC)-RIA and LC-multistage mass spectrometry. Two novel isomers of EO were elevated by Ang II; the second less polar isomer increased >50-fold in the A+F group. Central Ang II increased arterial expression of NCX1, TRPC6 and SERCA2 (2.6, 1.75 and 3.7-fold, respectively; P<0.01)) but not when co-infused with E or F. Adrenal and pituitary EO were unchanged. We conclude that brain Ang II activates a CNS-humoral axis involving plasma EO. The elevated EO reprograms peripheral ion transport pathways known to control arterial Na⁺ and Ca²⁺ homeostasis; this increases contractility and augments sympathetic effects. The new axis likely contributes to the chronic pressor effect of brain Ang II.     

A New Approach for the Modelling of Sediment Reworking Induced by a Macrobenthic Community

A new model of bioturbation has been developed to describe short term sediment reworking induced by macrobenthic communities. The design of the model had to consider the mixing processes, firstly, at the organism level, and secondly, at community level. This paper describes the mixing mode of the four types of bioturbators defined by the authors: the biodiffusors, the upward-conveyors, the downward-conveyors and the regenerators. The mathematical formulation of these sub-models consists of ordinary differential equations. They take into account the size of the bioturbated zone, the output fluxes to the water column, tracer decay, physical mixing due to local currents and the type and intensity of the bioturbation processes. These sub-models make it possible to describe correctly the mixing events that have occurred in cores with each type of bioturbator. They also provide the basis for general bioturbation model, that will take into account the respective degrees of involvement of (i) the different bioturbation processes and their characteristics, (ii) the interference between the different processes, and (iii) make possible to predict the particle reworking in order to include it in studies of organic matter in early diagenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Mucolytic Activity of Amides: A New Approach to Mucus Dispersion

A method which will reduce significantly the viscosity of epithelial mucus is essential to the physiological mechanisms involved in the mobilization and removal of such secretions. The life expectancy of patients with chronic pulmonary conditions and cystic fibrosis has been considerably increased and consequently the problem of liquefying mucin acquires new importance. In view of these considerations, as well as to facilitate research into the structural relationship of the glycoprotein macromolecule, a systematic investigation of mucolysis was undertaken using gastric mucin. Three amides, carbamide, acetamide and formamide, were found to dissolve gastric gel mucin with minimal degradation, and rapidly disperse the viscous secretions produced in pathological conditions of the tracheobronchial tree. Their effect on secretions from patients with cystic fibrosis and bronchiectasis is dramatic, and within five minutes of adding the reagent the flow time was reduced by at least 95%. Clinical studies were carried out with carbamide (urea in anhydrous, lyophilized, sterile powder form) in 32 patients with a variety of bronchial conditions, including chronic bronchitis, cystic fibrosis, asthma, bronchiectasis and emphysema. With the concentrations used, no irritant, bronchospastic or other reactions were observed.It is concluded that amides of this type have at least two actions on the epithelial mucous secretion: (1) breakage of the three-dimensional gel structure and (2) a slower reduction in viscosity followed by solution of the solid material.     

Using Biplanar Fluoroscopy to Guide Radiopaque Vascular Injections: A New Method for Vascular Imaging

Studying vascular anatomy, especially in the context of relationships with hard tissues, is of great interest to biologists. Vascular studies have provided significant insight into physiology, function, phylogenetic relationships, and evolutionary patterns. Injection of resin or latex into the vascular system has been a standard technique for decades. There has been a recent surge in popularity of more modern methods, especially radiopaque latex vascular injection followed by CT scanning and digital “dissection.” This technique best displays both blood vessels and bone, and allows injections to be performed on cadaveric specimens. Vascular injection is risky, however, because it is not a standardizable technique, as each specimen is variable with regard to injection pressure and timing. Moreover, it is not possible to view the perfusion of injection medium throughout the vascular system of interest. Both data and rare specimens can therefore be lost due to poor or excessive perfusion. Here, we use biplanar video fluoroscopy as a technique to guide craniovascular radiopaque latex injection. Cadaveric domestic pigs (Sus scrofa domestica) and white-tailed deer (Odocoileus virginianus) were injected with radiopaque latex under guidance of fluoroscopy. This method was found to enable adjustments, in real-time, to the rate, location, and pressure at which latex is injected in order to avoid data and specimen loss. In addition to visualizing the injection process, this technique can be used to determine flow patterns, and has facilitated the development of consistent markers for complete perfusion.     

A New Approach to Assess the Gastrocnemius Muscle Volume in Rodents Using Ultrasound; Comparison with the Gastrocnemius Muscle Index

Introduction

The purpose of this study was to determine the reliability and validity of a new non-invasive ultrasound technique to measure gastrocnemius muscle atrophy after nerve denervation in an animal model.

Methods

In sixteen rodents an eight mm sciatic nerve gap was created. In the following 8 weeks, each week, two rodents were euthanized and the gastrocnemius muscle was examined using two different ultrasound systems and two investigators. The standardized ultrasound measurement protocol consisted of identifying pre-defined anatomical landmarks: 1) the fibula, 2) the fibular nerve, and 3) the junction between the most distal point of the semitendinosus muscle and gastrocnemius muscle. Consequently, we measured the muscle thickness as the length of the line between the fibula and the junction between the two muscles, perpendicular to the fibular nerve. After the ultrasound recording, the muscle mass was determined.

Results

A steep decline of muscle weight of 24% was observed after one week. In the following weeks, the weight further decreased and then remained stable from 6 weeks onwards, resulting in a maximal muscle weight decrease of 82%. The correlation coefficient was >0.96 between muscle diameter and weight using both ultrasound systems. The inter-rater reliability was excellent for both devices on the operated side (ICC of 0.99 for both ultrasound systems) and good for the non-operated site (ICC’s: 0.84 & 0.89). The difference between the muscle mass ratio and the muscle thickness ratio was not more than 5% with two outliers of approximately 13%.

Discussion

We have developed an innovative, highly reliable technique for quantifying muscle atrophy after nerve injury. This technique allows serial measurements in the same animal over time. This is a significant advantage compared to the conventional technique for quantifying muscle atrophy, which requires sacrificing the animal.     

iTILLING: A Personalized Approach to the Identification of Induced Mutations in Arabidopsis

TILLING (for Targeting Induced Local Lesions IN Genomes) is a well-established method for identifying plants carrying point mutations in genes of interest. A traditional TILLING project requires a significant investment of time and resources to establish the mutant population and screening infrastructure. Here, we describe a modified TILLING procedure that substantially reduces the investment needed to perform mutation screening. Our motivation for developing iTILLING was to make it practical for individual laboratories to rapidly perform mutation screens using specialized genetic backgrounds. With iTILLING, M2 seeds are collected in bulk from the mutagenized population of plants, greatly reducing the labor needed to manage the mutant lines. Growth of the M2 seedlings for mutation screening, tissue collection, and DNA extraction are all performed in 96-well format. Mutations are then identified using high-resolution melt-curve analysis of gene-specific polymerase chain reaction products. Individual plants carrying mutations of interest are transferred from the 96-well growth plates to soil. One scientist can complete an iTILLING screen in less than 4 months. As a proof-of-principle test, we applied iTILLING to Arabidopsis (Arabidopsis thaliana) plants that were homozygous for the mekk1-1 (for MAPK/ERK kinase kinase 1) mutation and also carried a MEKK1 rescue construct. The goal of our screen was to identify mutations in the closely linked MEKK2 and MEKK3 loci. We obtained five mutations in MEKK2 and seven mutations in MEKK3, all located within 20 kb of the mekk1-1 T-DNA insertion. Using repeated iterations of the iTILLING process, mutations in three or more tandemly duplicated genes could be generated.The process of reverse genetics has been widely used by plant biologists to study gene function. In Arabidopsis (Arabidopsis thaliana), three approaches that have been used to generate populations of plants for reverse genetic analysis are insertional mutagenesis (Wisman et al., 1998; Alonso et al., 2003), fast neutron mutagenesis to induce deletions (Li et al., 2001), and chemical mutagenesis to induce point mutations (McCallum et al., 2000). In order to find individual plants carrying point mutations of interest, a process called TILLING (for Targeting Induced Local Lesions IN Genomes) was developed whereby genes are screened for mutations using a PCR-based assay (McCallum et al., 2000). Although originally developed for use with Arabidopsis, the TILLING process has been subsequently applied to a wide range of plants, including barley (Hordeum vulgare; Caldwell et al., 2004), Brassica napus (Wang et al., 2008), Brassica oleracea (Himelblau et al., 2009), Brassica rapa (Stephenson et al., 2010), Lotus japonicus (Perry et al., 2009), maize (Zea mays; Till et al., 2004), Medicago truncatula (Le Signor et al., 2009), oat (Avena sativa; Chawade et al., 2010), pea (Pisum sativum; Triques et al., 2007), potato (Solanum tuberosum; Elias et al., 2009), rice (Oryza sativa; Till et al., 2007), sorghum (Sorghum bicolor; Xin et al., 2008), soybean (Glycine max; Cooper et al., 2008), tomato (Solanum lycopersicum ; Gady et al., 2009), and wheat (Triticum aestivum; Dong et al., 2009). TILLING has also been used in Drosophila (Winkler et al., 2005), zebrafish (Wienholds et al., 2003), and Caenorhabditis elegans (Gilchrist et al., 2006).The chemical mutagen most commonly used to create the mutant populations used for TILLING is ethyl methanesulfonate (EMS). When working with plants, seeds are soaked in EMS to induce mutations throughout the genome. Mutagenized seeds are then planted on soil, and the resulting plants are grown to maturity to produce M2 seeds, which are collected from the plants individually or in small pools. Next, M2 seed samples from each individual plant are germinated and grown to produce tissue from which DNA can be extracted. The resulting large collection of ordered DNA samples and the corresponding M2 seeds constitute the infrastructure of a TILLING population. PCR-based screening can then be used to find individual plants in the population carrying mutations in genes of interest (McCallum et al., 2000). Once established, this type of TILLING infrastructure can serve the needs of an entire research community through a fee-for-service screening operation (Colbert et al., 2001; Martín et al., 2009).Several different strategies have been developed for identifying the mutations present in a TILLING population, but all of them involve detecting heteroduplex PCR products. A heteroduplex is formed when a mixture of wild-type and mutant PCR products are melted and reannealed, resulting in DNA duplexes that contain a single-base mismatch. TILLING was originally described using denaturing HPLC to identify mutations based on the differential retention times of heteroduplexes and homoduplexes in the chromatography column (McCallum et al., 2000). TILLING has since been modified so that endonucleases are used to cleave PCR products containing a heteroduplex. Cleavage products are then separated via gel electrophoresis to identify banding patterns indicative of mutations (Colbert et al., 2001).More recently, high-resolution melting analysis of PCR products has been used to identify heteroduplexes when performing TILLING (Dong et al., 2009; Gady et al., 2009). High-resolution melting analysis was originally developed for use in clinical settings to identify known single-nucleotide polymorphisms and small insertions/deletions potentially linked to genetic diseases (Erali et al., 2008). With high-resolution melting, the mismatch in a heteroduplex is visualized as a melting event that occurs more rapidly or at a lower temperature than the corresponding homoduplex. Montgomery et al. (2007) demonstrated that mutation scanning with high-resolution melting is a robust technique with greater than 95% sensitivity in distinguishing heteroduplexes from homoduplexes. It has also been observed that the sensitivity with which mutations in PCR products can be identified using DNA melting analysis depends on the resolution of the instrumentation used for collecting the melt-curve data (Zhou et al., 2005; Herrmann et al., 2006).Although traditional TILLING is a high-throughput method for mutation screening, the establishment of the initial screening population and the corresponding ordered DNA samples requires a substantial up-front investment of time and money. Because of this situation, TILLING resources are available for only two genetic backgrounds in Arabidopsis: wild-type Columbia-0 and Landsberg erecta (Greene et al., 2003; Martín et al., 2009). If a scientist is interested in identifying mutations in a more specialized genetic background, the costs associated with establishing a TILLING population can be prohibitive. Therefore, we were interested in determining if a modified version of the TILLING process could be developed that would substantially reduce the investment of time and resources necessary to perform mutation screening. The individualized TILLING procedure, or iTILLING, which we describe in this paper provides one solution to this challenge.     

Diving-for-Food: A New Model to Assess Social Roles in a Group of Laboratory Rats

In an experimental situation called “diving for food”, groups of laboratory rats are tested in an aquarium where they have to dive and swim under water to reach the single source of food located at the other end. A behavioural differentiation appears: some rats — the carriers — dive to get food and others — non-carriers — stay in the cage and feed by stealing. We examine whether carrier and non-carrier profiles can be considered as social roles, defined as supraindividual features dependent on the social context. Carrier/non-carrier differentiation resulted in all groups tested. Individually tested, almost all rats can get food by diving and swimming. Differentiation also occurred in groups that had been previously trained alone in the device, and in groups whose members had all been carriers or non-carriers exclusively in a preliminary stage. As the access-to-food behaviour of a rat having to cope with the diving-for-food situation is settled by its social environment, we consider that the present experimental model is promising for the study of interactions between the individual and the social structure.     

PolNet: A Tool to Quantify Network-Level Cell Polarity and Blood Flow in Vascular Remodeling

In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.     

A Bayesian MCMC Approach to Assess the Complete Distribution of Fitness Effects of New Mutations: Uncovering the Potential for Adaptive Walks in Challenging Environments

The role of adaptation in the evolutionary process has been contentious for decades. At the heart of the century-old debate between neutralists and selectionists lies the distribution of fitness effects (DFE)—that is, the selective effect of all mutations. Attempts to describe the DFE have been varied, occupying theoreticians and experimentalists alike. New high-throughput techniques stand to make important contributions to empirical efforts to characterize the DFE, but the usefulness of such approaches depends on the availability of robust statistical methods for their interpretation. We here present and discuss a Bayesian MCMC approach to estimate fitness from deep sequencing data and use it to assess the DFE for the same 560 point mutations in a coding region of Hsp90 in Saccharomyces cerevisiae across six different environmental conditions. Using these estimates, we compare the differences in the DFEs resulting from mutations covering one-, two-, and three-nucleotide steps from the wild type—showing that multiple-step mutations harbor more potential for adaptation in challenging environments, but also tend to be more deleterious in the standard environment. All observations are discussed in the light of expectations arising from Fisher’s geometric model.     

A Novel Approach to Recovery of Function of Mutant Proteins by Slowing Down Translation

Protein homeostasis depends on a balance of translation, folding, and degradation. Here, we demonstrate that mild inhibition of translation results in a dramatic and disproportional reduction in production of misfolded polypeptides in mammalian cells, suggesting an improved folding of newly synthesized proteins. Indeed, inhibition of translation elongation, which slightly attenuated levels of a copepod GFP mutant protein, significantly enhanced its function. In contrast, inhibition of translation initiation had minimal effects on copepod GFP folding. On the other hand, mild suppression of either translation elongation or initiation corrected folding defects of the disease-associated cystic fibrosis transmembrane conductance regulator mutant F508del. We propose that modulation of translation can be used as a novel approach to improve overall proteostasis in mammalian cells, as well as functions of disease-associated mutant proteins with folding deficiencies.     

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λP_R or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.