Integrative taxonomy recognized a new cryptic species within Stipa grandis from Loess Plateau of China

Stipa shanxiensis, a cryptic species within Stipa grandis that originated from central and western China, is described based on morphological, genomic, and ecological data from field and common garden experiments. Stipa shanxiensis morphologically resembles S. grandis, although phylogenetically it is closely related to the less morphologically similar Stipa baicalensis and Stipa krylovii. Of the eight significant morphological differences between S. shanxiensis and S. grandis, the two, cauline ligules longer than 2 cm with a filiform apex, and hairs shorter than 0.2 mm on the adaxial surface of the cauline uppermost leaves can be used to distinguish the species. Results from a common garden experiment verified that the two diagnostic characteristics were relatively stable and less morphologically plastic in response to environmental variation. Furthermore, a significant ecological divergence was found between S. shanxiensis and S. grandis, such that the former preferred warmer and more humid climates, and their predicted distribution was generally separated. Taken together, our results highlight that the integrative taxonomic approach was valuable for recognizing a new cryptic species in Stipa. In particular, we find that common garden experiments involving the effects of growth stage and characteristic position helped to morphologically diagnose cryptic species. These findings may also facilitate our understandings of ecological adaption and phenotypic plasticity in response to environmental change.     

矮珍珠硝酸盐转运蛋白基因GeNRT2.1的克隆和功能研究

硝酸盐转运蛋白（nitrate transporter,NRT）是植物识别、吸收和转运硝酸盐的关键蛋白,对促进作物根系发育、提高产量具有重要作用。通过筛选水生植物,利用NRT蛋白的保守区设计简并引物,并通过PCR和RACE技术,首次从矮珍珠（Glossostigma elatinoides）中克隆得到GeNRT2.1基因。进化分析结果表明,GeNRT2.1与烟草NRT2.1在进化关系上距离最近。qRT-PCR结果表明,GeNRT2.1在矮珍珠根中表达量最高,其次是叶和茎,此外,低浓度硝酸盐（0.5 mmol·L^-1）处理后,GeNRT2.1在根、叶、茎中的表达量分别是高浓度硝酸（2 mmol·L^-1）处理后的1.89、1.93和2.07倍。功能互补实验发现,GeNRT2.1能使缺陷型酵母Δynr恢复生长,具有硝酸盐转运蛋白的功能。通过丰富NRT基因资源,以期为培育氮肥高效利用转基因作物,发展绿色农业,保证我国的粮食安全和环境安全提供理论依据。     

Optimization of fed-batch parameters and harvest time of CHO cell cultures for a glycosylated product with multiple mechanisms of inactivation

Optimization of fed-batch feeding parameters was explored for a system with multiple mechanisms of product inactivation. In particular, two separate mechanisms of inactivation were identified for the recombinant tissue-type activator (r-tPA) protein. Dynamic inactivation models were written to describe particular r-tPA glycoform inactivation in the presence and absence of free-glucose. A glucose-independent inactivation mechanism was identified, and inactivation rate constants were found dependent upon the presence of glycosylation of r-tPA at N184. Inactivation rate constants of the glucose-dependent mechanism were not affected by glycosylation at N184. Fed-batch optimization was performed for r-tPA production by CHO cell culture in a stirred-tank reactor with glucose, glutamine and asparagine feed. Feeding profiles in which culture supernatant concentrations of free-glucose and amino acids (combined glutamine and asparagine) were used as control variables, were evaluated for a wide variety of set points. Simulation results for a controlled feeding strategy yielded an optimum at set points of 1.51 g L(-1) glucose and 1.18 g L(-1) of amino acids. Optimization was also performed in absence of metabolite control using fixed feed-flow rates initiate during the exponential growth phase. Fixed feed-flow results displayed a family of optimum solutions along a mass flow rate ratio of 3.15 of glucose to amino acids. Comparison of the two feeding strategies showed a slight advantage of rapid feeding at a fixed flow rate as opposed to metabolite control for a product with multiple mechanisms of inactivation.     

Factors affecting protein refolding yields in a fed-batch and batch-refolding system

The refolding of recombinant protein from inclusion bodies expressed in Escherichia coli can present a process bottleneck. Yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. This article studies the effect of five factors upon the dilution refolding of protein in a twin impeller fed-batch system using refold buffer containing only the oxidized form of the redox reagent. Such a buffer is easier to prepare and more stable than a buffer containing both reduced and oxidized forms. The five factors chosen were: bulk impeller Reynolds number, mini-impeller Reynolds number, injection rate of denatured protein, redox ratio, and guanidine hydrochloride (GdHCl) concentration. A 2(5) factorial experiment was conducted at an industrially relevant protein concentration using lysozyme as the test system. The study identified that in the system used, the guanidine hydrochloride concentration, redox ratio, and injection rate were the most important factors in determining refolding yields. Two interactions were found to be important: redox ratio/guanidine hydrochloride concentration and guanidine hydrochloride concentration/injection rate. Conditions were also found at which high refolding yields could be achieved even with rapid injection and poor mixing efficiency. Therefore, a comparative assessment was carried out with minimal mixing in a simple batch-refolding mode of operation, which revealed different behavior to that of fed-batch. A graphical (windows of operation) analysis of the batch data suggested that optimal yields and productivity are obtained at high guanidine hydrochloride concentrations (1.2 M) and redox ratios of unity or greater.     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

Respirometric evaluation and modeling of glucose utilization by Escherichia coli under aerobic and mesophilic cultivation conditions

The study presents a mechanistic model for the evaluation of glucose utilization by Escherichia coli under aerobic and mesophilic growth conditions. In the first step, the experimental data was derived from batch respirometric experiments conducted at 37 degrees C, using two different initial substrate to microorganism (S(0)/X(0)) ratios of 15.0 and 1.3 mgCOD/mgSS. Acetate generation, glycogen formation and oxygen uptake rate profile were monitored together with glucose uptake and biomass increase throughout the experiments. The oxygen uptake rate (OUR) exhibited a typical profile accounting for growth on glucose, acetate and glycogen. No acetate formation (overflow) was detected at low initial S(0)/X(0) ratio. In the second step, the effect of culture history developed under long-term growth limiting conditions on the kinetics of glucose utilization by the same culture was evaluated in a sequencing batch reactor (SBR). The system was operated at cyclic steady state with a constant mean cell residence time of 5 days. The kinetic response of E.coli culture was followed by similar measurements within a complete cycle. Model calibration for the SBR system showed that E. coli culture regulated its growth metabolism by decreasing the maximum growth rate (lower microH) together with an increase of substrate affinity (lower K(S)) as compared to uncontrolled growth conditions. The continuous low rate operation of SBR system induced a significant biochemical substrate storage capability as glycogen in parallel to growth, which persisted throughout the operation. The acetate overflow was observed again as an important mechanism to be accounted for in the evaluation of process kinetics.     

血立停胶囊对早孕大鼠RU486药流后子宫收缩的实验研究

目的:研究血立停胶囊对早孕大鼠Ru486药物流产后对子宫平滑肌收缩频率、收缩幅度及活动力变化的影响,旨在探讨血立停胶囊治疗药物流产后出血的作用机制。方法:选择妊娠Wistar大鼠,随机分为6组,即对照组,米非司酮组,大剂量血立停组,小剂量血立停组,催产素组,止血敏组。于妊娠第7天,开始相应处理,妊娠第14天分别监测以下指标后处死:在体子宫平滑肌收缩力,收缩幅度、收缩频率。结果:大剂量血立停可明显增强大鼠在体子宫平滑肌活动力、提高子宫平滑肌收缩频率、收缩幅度,与对照组比较差异有显著性(p<0.05)。结论:血立停胶囊可增强大鼠子宫平滑肌兴奋性,从而起到对药物流产后阴道出血的治疗作用。     

Effect of physiological heterogeneity of E. coli popula-tion on antibiotic susceptivity test

According to the instantaneous growth rate (dN/dt) of E. coli CVCC249 growing in batch culture, the entire growth progress was distinguished into four phases: accelerating growth phase, constant growth phase, decelerating growth phase and declining phase, in each of which obvious variation in physiological and biochemical properties was detected, including total DNA, total protein, and MTT-dehydrogenase activity, etc., that led to difference in their antibiotic susceptivity. Antibiotic susceptivity of the population sampled from each phase was tested by Concentration-killing Curve (CKC) approach following the formula N=N ₀/{1+exp[r·(x-BC ₅₀)]}, showing as normal distribution at the individual cell level for an internal population, in which the median bactericidal concentration BC ₅₀ represents the mean level of susceptivity, while the bactericidal span BC ₁₋₉₉=(2lnN ₀)/r indicates the variation degree of the antibiotic susceptivity. Furthermore, tested by CKC approach, the antibiotic susceptivity of E. coli CVCC249 population in each physiological phase to gentamicin or enoxacin was various: susceptivity of the population in the constant growth phase and declining phase all increased compared with that in the accelerating growth phase for gentamicin but declined for enoxacin. The primary investigations revealed that the physiological phase should be taken into account in the context of antibiotic susceptivity and research into antimicrobial mechanism. However there are few reports concerned with this study. Further research using different kinds of antibiotics with synchronized continuous culture of different bacterial strains is required. Supported by the Natural Science Foundation of Shandong Province, China (Grant No. Y2005C58), the Natural Key Technology R&D Program of China (Grant No. 2006BAK02A03-6) and the Youth Scientific Research Foundation of Shandong Academy of Agricultural Science (2005YQ035)