Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.     

The crystal structure of the sorcin calcium binding domain provides a model of Ca2+-dependent processes in the full-length protein

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH.The sorcin calcium binding domain (SCBD, residues 33-198) expressed in Escherichia coli was crystallized in the Ca2+-free form. The structure was solved by molecular replacement and was refined to 2.2 A with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers.The structure of the SCBD leads to a model that explains the solution properties and describes the Ca2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca2+-dependent binding to target proteins at or near membranes.     

脉冲电场下肿瘤细胞的窗口效应

基于多层电介质模型,对于适应于球形生物细胞的脉冲电场,提出了一种等效电路模型,在相同频域下,内膜和外膜的变化趋势相同,频域分析表明,不同频谱场将引起不同的生物医学效应．我们针对癌症细胞计算了跨膜电压,并讨论了脉冲和跨膜电压以及阻抗的关系．结果表明不同的频域和不同的持续时间对细胞的内膜和外膜有选择性的影响,时域和频域的分析显示,在细胞上有一个窗口,当持续时间在10^-8～10^-6 s之间,细胞内膜的电压将高于细胞外膜的电压．窗口效应为解释生物细胞的脉冲电学效应提供了一种参考思路．     

Crystal structure of calcium-free human sorcin: A member of the penta-EF-hand protein family

Sorcin is a 22 kD calcium-binding protein that is found in a wide variety of cell types, such as heart, muscle, brain and adrenal medulla. It belongs to the penta-EF-hand (PEF) protein family, which contains five EF-hand motifs that associate with membranes in a calcium-dependent manner. Prototypic members of this family are the calcium-binding domains of calpain, such as calpain dVI. Full-length human sorcin has been crystallized in the absence of calcium and the structure determined at 2.2 A resolution. Apart from an extended N-terminal portion, the sorcin molecule has a globular shape. The C-terminal domain is predominantly alpha-helical, containing eight alpha-helices and connecting loops incorporating five EF hands. Sorcin forms dimers through the association of the unpaired EF5, confirming this as the mode of association in the dimerization of PEF proteins. Comparison with calpain dVI reveals that the general folds of the individual EF-hand motifs are conserved, especially that of EF1, the novel EF-hand motif characteristic of the family. Detailed structural comparisons of sorcin with other members of PEF indicate that the EF-hand pair EF1-EF2 is likely to correspond to the two physiologically relevant calcium-binding sites and that the calcium-induced conformational change may be modest and localized within this pair of EF-hands. Overall, the results derived from the structural observations support the view that, in sorcin, calcium signaling takes place through the first pair of EF-hands.     

Fermentation at non-conventional conditions in food- and bio-sciences by the application of advanced processing technologies

The interest in improving the yield and productivity values of relevant microbial fermentations is an increasingly important issue for the scientific community. Therefore, several strategies have been tested for the stimulation of microbial growth and manipulation of their metabolic behavior. One promising approach involves the performance of fermentative processes during non-conventional conditions, which includes high pressure (HP), electric fields (EF) and ultrasound (US). These advanced technologies are usually applied for microbial inactivation in the context of food processing. However, the approach described in this study focuses on the use of these technologies at sub-lethal levels, since the aim is microbial growth and fermentation under these stress conditions. During these sub-lethal conditions, microbial strains develop specific genetic, physiologic and metabolic stress responses, possibly leading to fermentation products and processes with novel characteristics. In some cases, these modifications can represent considerable improvements, such as increased yields, productivities and fermentation rates, lower accumulation of by-products and/or production of different compounds. Although several studies report the successful application of these technologies during the fermentation processes, information on this subject is still scarce and poorly understood. For that reason, the present review paper intends to assemble and discuss the main findings reported in the literature to date, and aims to stimulate interest and encourage further developments in this field.     

Circadian variability in airways characteristics: A spirometric study

ABSTRACT

Background: Chronic obstructive pulmonary disease (COPD) and Asthma patients exhibit exacerbation of symptoms in night hours and early morning. Temporal variability in airway caliber have been reported in past using peak expiratory flow rate which represents large airways caliber, while in COPD and Asthma, smaller airways are particularly affected. We studied circadian variability of airway caliber using Forced Expiratory Volume in the First Second (FEV1) and Mid Expiratory Flow rate.

Methods: Male volunteers (18–26 years), having similar daily routine were recruited. Spirometry was performed at 5: 00, 8:00, 11:00, 14:00, 17:00, 20:00 and 23:00 h. Data from 104 subjects was analyzed for diurnal variability parameters viz., amplitude percent mean and standard deviation percent of mean. For circadian rhythm Cosinor curve was fitted and rhythm characteristics in terms of MESOR, Amplitude and Acrophase were determined.

Results: Repeated measures ANOVA revealed significant differences in spirometric parameters measured at different time points during the day. In general, spirometric parameters follow a sinusoidal pattern and exhibit minimum values during night hours and maximum values during day time. FEV1 Cosinor rhythm was significant in 31% of subjects (Zero amplitude test). The distribution of acrophase revealed interindividual differences in chronophenotypes. Variability was minimum for FEV1% and maximum for FEF75 suggesting dynamic interplay of airway geometry and neuro-chemical influences.

Conclusion: The presence of different chronophenotypes in normal subjects suggests that the nocturnal asthma may also be a different phenotype. Availability of portable spirometers and home monitoring thus may be required for ascertaining chronophenotype and tailoring chronotherapeutic interventions.     

Calpains — An elaborate proteolytic system

Calpain is an intracellular Ca²⁺-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca²⁺via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

猪血红蛋白(Hb)脉冲酶解效果比较研究

目的寻找一种高效快捷有效地降解猪血红蛋白(Hb)新方法。方法在波型为双向方波,电极间距离为1.2 cm,脉冲频率为200 kHz的脉冲电场下,利用胰蛋白酶在温度为37℃,水解时间为4 h条件下水解猪血红蛋白。结果在脉冲电场作用下,胰蛋白酶水解血红蛋白获得的降解产物,利用高效凝胶色谱、紫外可见扫描及SDS-PAGE蛋白质电泳检测,发现其吸收峰或色带明显多于单一利用胰蛋白酶降解血红蛋白所得降解产物的吸收峰或色带。结论当脉冲电场通过血红蛋白时,血红蛋白内部的分子结构便产生斯塔克效应(Stark effect),引起血红蛋白分子剧烈振动,从而改变其分子结构振辐、吸收峰和偶极矩,并分别引起斯塔克频率、偶极矩、极化率的改变、使血红蛋白分子结构的极化跃迁和超极化,因此,在脉冲电场作用下,促进了血红蛋白酶解反应。     

ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.     

Mechanisms involved in the antinociception of petroleum ether fraction from the EtOH extract of Chrysanthemum indicum in mice

The petroleum ether fraction (PEF) from the EtOH extract of flowers and buds of Chrysanthemum indicum was evaluated on antinociception in mice using chemical and thermal models of nociception. PEF administered orally at doses of 188 and 376 mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid, subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and the hot plate test. In the pentobarbital sodium-induced sleep time test and the open-field test, PEF neither enhanced the pentobarbital sodium-induced sleep time nor impaired the motor performance, indicating that the observed antinociception was unrelated to sedation or motor abnormality. In a measurement of core body temperature, PEF did not affect temperature within 80 min. Moreover, PEF-induced antinociception in the capsaicin test was insensitive to naloxone, yohimbine or methylene blue, but was significantly antagonized by atropine and glibenclamide. These results suggested that PEF-produced antinociception might be involvement in the ATP sensitive K+ channels and the mAChRs-ATP sensitive K+ channels pathway. In additional, the antinociception of PEF might attribute to the synergic effects of these two compounds, 2-[[2-[2-[(2-ethylcyclopropyl)methyl] cyclop Cyclopropaneoctanoic and n-hexadecanoic acid, or the property of a single compound, which merited exploring further.