Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium

The lipid emulsion components of a serum-free insect cell medium were varied and evaluated for effects on cell growth and recombinant protein expression. The growth of High-Five^TM cells was significantly affected by polyol Pluronic F-68 and Tween-80, but not by lipids. Pluronic was essential for cell growth, while Tween-80 was required to achieve maximum cell densities. A dose response effect was observed for Tween-80 with optimal cell growth at a concentration of 25 mg/l. Cholesterol had a minor effect on cell growth, but was essential for the expression of recombinant proteins. The expression of -galactosidase (-gal) was directly affected by cholesterol with optimal expression at a concentration of 5.4 mg/l. Vitamin E, important as an antioxidant to stabilize lipids, did not directly affect recombinant protein expression. Although lipids were not required for cell growth, the presence of lipids were required during the cell growth phase in order to achieve efficient infection with baculovirus. These studies help to define the important components, and range of concentrations, for lipid emulsions which can effectively replace serum in insect cell culture.Abbreviations -gal galactosidase (E.C. 3.2.1.23) - Sf-9 Spodoptera frugiperda - High-5 Trichoplusia ni 5Bl-4     

Optimization of γ-linolenic acid (GLA) production inSpirulina platensis

The cyanobacteriumSpirulina platensis is one of the most promising sources of the polyunsaturated fatty acid -linolenic acid (GLA). The GLA content ofSpirulina can be enhanced by cultivation under light-dark cycles in the laboratory or outdoors. Thus, in strain BP, the GLA content increased from 1.2 to 1.6% when cultivated under light-dark cycles. Moreover, in the derived mutant Z19, the GLA content reached 2.4% when cultivated outdoors. To the best of our knowledge, this is the highest GLA content ever reported for any alga.Author for correspondence     

Peculiar behavior of the nucleolus and appearance of cytoplasmic nucleolus-like bodies in the roottip meristems ofBrodiaea uniflora Engl. Grown at low temperature

Summary Cytoplasmic nucleolus-like bodies, which showed vast variation both in size and in number per cell, were sometimes found in the telophase cells ofBrodiaea uniflora. When the plants were grown at low temperature, the frequency of telophase cells bearing cytoplasmic nucleolus-like bodies increased with the lapse of days. In contrast, growth at moderate temperature reduced this frequency. Prolonged exposure of the plants to low temperature caused peculiar phenomena concerning the behavior of the nucleolus and nucleolar material: 1. retention of nucleolar remnants at high frequency in metaphase, 2. pulverization of the nucleolar remnants into a great number of minute, fluffed fragments during metaphase, 3. appearance of dot-like nucleolar material in anaphase, and 4. appearance of nucleolus-like bodies, sometimes more than 10 m in diameter, in telophase. All these structures were strongly impregnated with silver. Electron microscopy revealed that both the nucleolar remnant and the nucleolus-like body consisted primarily of fibrils. Our observations clearly demonstrate that the nucleolus-like bodies are derived from the fibrillar component of the nucleolus and are formed by fusion of dot-like nucleolar material during anaphase.     

Methylation of progesterone receptor isoform A promoter in normal breast tissue: An epigenetic link between early age at menarche and risk of breast cancer?

Emerging evidence indicates that some altered patterns of methylation that occur in breast tumors may also be found in breast tissue of healthy women in relation to the breast cancer (BC) risk factors. Progesterone receptor (PR) isoform α is a crucial regulator of breast hormone responsiveness and its hypermethylation plays an important role in the initiation and development of breast tumors. However, such a methylation change in healthy women and its link with the different risk factors has not yet been investigated. In the present study, we aimed to examine the relationship of possible methylation changes within a critical region in the promoter CpG island of PGR-α (progesterone receptor α) gene in the healthy women with a set of reproductive and nonreproductive BC risk factors. The breast tissues were collected from 120 cancer-free women who had undergone cosmetic mammoplasty. The genomic DNA was extracted from the breast tissues and the methylation level of PGR-α promoter CpG island was determined by using MeDIP-qPCR assay. Using regression analysis, we found that increasing menarche age is inversely associated with the high methylation of PGR-α promoter ( β = −0.790, SE = 0.362; P = 0.031). Although lactating women had more methylation than nonlactating women (P = 0.026, the t test), this result was not confirmed by regression models. Such an observation may be helpful in better understanding of the underlying mechanisms by which early age at menarche increases the risk of BC. However, this perspective requires further validations in larger studies of more subjects as well as the inclusion of other related genes.     

Differential inhibition of regional gastrointestinal tissue to progesterone in the rat

The influence of progesterone on contractile activity of three gastrointestinal regional tissues was evaluated. Up to six dose levels of progesterone were administered subcutaneously to male rats daily for four days. Progesterone blood levels measured with radioimmunoassay on the fourth day revealed that the range of progesterone exposure to the male animals did not exceed the progesterone blood level peak reported during the normal hormonal cycle of female rats. Log-dose response curves indicate that esophageal, antral, and colonic tissues from progesterone treated animals showed a significant reduction in contractile activity compared to the corresponding tissue from non-progesterone treated control animals. Esophageal and colonic tissues were two and 12-fold, respectively, more sensitive to the inhibitory progesterone influence compared to antral tissue. This study supports the concept that normal circulating levels of progesterone may have an influence on specific gastrointestinal regional function in addition to the effects of progesterone blood levels during pregnancy.     

Antioxidant and antimicrobial potential of two extracts from Capparis spinosa L. and Rumex nervosus and molecular docking investigation of selected major compounds

The present study examined the phytochemical composition, antioxidant, antimicrobial properties, and molecular docking of different solvents extracts (methanol and water) of two medicinal plants, namely, Capparis spinosa L (CS) and Rumex nervosus (RN). Phytochemical analysis showed that total phenol, flavonoids, alkaloids, and vitamin C were significantly (P ≤ 0.05) higher in the methanolic extract of both plants than in other solvents. However, tannin content was significantly (P ≤ 0.05) high in the water extract for both plants. Chloroform and acetone extracts were significantly lower in phytochemicals than other solvents, therefore excluded in this study. GC–MS analysis showed one dominant compound in CS (isopropyl isothiocyanate) and two in RN (pyrogallol and palmitic acid). The antioxidant methods applied (DPPH, ABTS, β-Carotene/linoleic acid assay, and reducing the power) showed that the methanolic extract of CS exerted higher activity in methanolic extract but lower than that of BHA standard. The methanolic extract of both plants inhibited the bacterial pathogens when a minimum inhibitory concentration (MIC) method was applied, compared to water extract with RN-methanolic extract had a lower inhibition concentration than CS-methanolic extract. The molecular interactions study revealed that the palmitic acid and pyrogallol interacted with the receptors' active site. This work concluded that CS and RN showed a remarkable antioxidant and antibacterial effect with the high antimicrobial activity of RN extract.     

短链壬基酚聚氧乙烯醚脱氢酶脱氢催化机理

【目的】为研究短链壬基酚聚氧乙烯醚脱氢酶(sNPEO-DH)的脱氢氧化机制(基因克隆于Ensifer sp.AS08),我们进行了以下实验。【方法】采用同源序列比对及同源建模的方法筛选出与其辅酶黄素腺嘌呤二核苷酸(FAD)异咯嗪基邻近的4个氨基酸残基。以定点突变方法分别构建了突变体,并进行了重组蛋白的表达纯化和酶活力测定。【结果】野生型和突变体的酶学动力学实验表明,突变体N90A和N509A对亲水性底物聚乙二醇(PEG1000)的相对活性分别降低为51%和89%,对疏水性底物sNPEO的活性分别降低为26%和40%,说明氨基酸残基N90和N509可能与底物的结合相关。突变体H465A的相对活性丧失了90%以上,突变体N507A完全丧失活性;瞬时"停-流"检测实验进一步证明N507A突变体阻断了底物向FAD传递质子的过程,突变体H465A阻断了对FAD还原形成的FADH2脱氢再生的过程。【结论】以上结果说明N507和H465为sNPEO脱氢酶活性中心中参与对底物氧化脱氢及FADH2脱氢再生进行下一次反应的催化位点。     

PGR-08理化性质及生物活性的研究

PGR-0 8是从某种土壤放线菌 (Actinomyces)的发酵液中分离提纯得到的白色结晶 ,纸层析和熔点检测证明它是化学单体 ,mp 1 80～ 1 81℃ ,[α]2 1D+ 4 3°(H2 O)。溶解性试验表明它易溶于 H2 O(≥ 6 0℃ )、HCl、Na OH、(CH3 ) 2 CO、CH3 OH和 C2 H5OH等 ,但不溶于 Na HCO3 。Tollen、Fehling和 Ag NO3 -溴酚兰等专性试剂的颜色反应表明其组成成分中有嘌呤和糖。生物试验证明 :PGR-0 8具有细胞分裂素的活性 ,例如它能减缓蒲公英离体叶圆片 Chl.的降解 ,延长叶圆片寿命 1～ 2倍以上 ,诱导叶圆片分化出不定根或不定芽 ,甚至建成小植株。它还能促进小麦种子萌发。     

Evolution of miniaturisation in inquiline parasitic ants: Timing of male elimination in <Emphasis Type="Italic">Plagiolepis pygmaea</Emphasis>, the host of <Emphasis Type="Italic">Plagiolepis xene</Emphasis>

Let v be a valuation of terms of type , assigning to each term t of type a value v(t) 0. Let k 1 be a natural number. An identity of type is called k-normal if either s = t or both s and t have value k, and otherwise is called non-k-normal. A variety V of type is said to be k-normal if all its identities are k-normal, and non-k-normal otherwise. In the latter case, there is a unique smallest k-normal variety to contain V , called the k-normalization of V. Inthe case k = 1, for the usual depth valuation of terms, these notions coincide with the well-known concepts of normal identity, normal variety, and normalization of a variety. I. Chajda has characterized the normalization of a variety by means of choice algebras. In this paper we generalize his results to a characterization of the k-normalization of a variety, using k-choice algebras. We also introduce the concept of a k-inflation algebra, and for the case that v is the usual depth valuation of terms, we prove that a variety V is k-normal iff it is closed under the formation of k-inflations, and that the k-normalization of V consists precisely of all homomorphic images of k-inflations of algebras in V .     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.