Mammalian cell protein expression for biopharmaceutical production

Mammalian cell expression has become the dominant recombinant protein production system for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly. While expression and production have been fully developed and Chinese hamster ovary cells are used for the majority of products both on the market and in clinical development, significant progresses in developing and engineering new cell lines, introducing novel genetic mechanisms in expression, gene silencing, and gene targeting, have been reported in the last several years. With the latest analytical methods development, more attention is being devoted towards product quality including glycol profiling, which leads to better understanding the impact of culture condition during production. Additionally, transient gene expression technology platform plays more important role in biopharmaceutical early development stages. This review focused on the latest advancements in the field, especially in active areas such as expression systems, glycosylation impact factors, and transient gene expression.     

Inflammation factors and element supplementation in cancer

The aim of the study was to evaluate the effect of dietary supplementation with chosen minerals (Zn, Se, Fe) on expression of selected cytokines (IL-1, IL-6, TNFα) in spleen of rats and on their concentrations in rat serum under inflammatory and pathological conditions obtained by implantation of prostate cancer cells (LnCaP). Serum levels of metabolites of arachidonic, eicosapentaenoic and linoleic acids (hydroxyeicosatetraenoic, hydroxyeicosapentaenoic and hydroxyoctadecadienoic acids, respectively), as compounds involved in inflammation and cancer development, were also investigated. Male rats were randomised into dietary groups supplemented with Zn, Se or Fe. Prostate cancer cells were implanted to some rats in each group.The study demonstrated that minerals supplemented with the diet may exert various effects on an organism. Selenium, zinc and iron influence pro-inflammatory cytokine expression, what leads to stimulation of inflammation. They also affect synthesis of arachidonic and linoleic acid metabolites that exert pro-inflammatory action and enable cancer development and metastasis.     

Concentration of selected elements in the roots and crowns of both primary and permanent teeth with caries disease

We examined the differences between the concentrations of chemical elements in caries-changed primary teeth and permanent ones with a division into the root and the crown. The study comprised 27 children aged from 4 to 11 yr and 36 adults aged from 36 to 71 yr. We examined the elements with the total reflection X-ray fluorescence method. The lowest concentrations of calcium, manganese, strontium, lead, and copper were in the roots of primary teeth compared to the roots of permanent ones. The calcium, nickel, zinc, lead, and copper concentrations were significantly higher in the roots of primary teeth than in the roots of permanent teeth. However, the zinc concentration was higher both in the root and crown of primary teeth than in permanent teeth. On the basis of our investigations, we can conclude that the content of some elements (manganese, copper, strontium, and lead) is higher in caries permanent teeth than in primary ones. The nickel and zinc concentrations are higher in the teeth of the children than the adults. However, the content of other elements (calcium, chromium, iron) is similar in both kinds of teeth.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Distribution of elements binding to molecules with different molecular weights in aqueous extract of Antarctic krill by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000-20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000-20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.     

镱和铕诱导金属硫蛋白生成的初步研究

本文研究了不同剂量的稀土元素镱和铕,通过皮下注射对兔肝中金属硫蛋白生成的影响。用ＳｅｐｈａｄｅｘＧ５０柱对兔肝提取液进行分离,收集的组分用紫外分光光度法、原子吸收光谱法和ＥＬＩＳＡ法进行分析。实验结果表明,低剂量（１－３ｐｐｍ）的镱和铕不能明显地诱导兔肝内金属硫蛋白的合成,而高剂量（１０－４０ｐｐｍ）的镱和铕可以诱导兔肝金属硫蛋白合成。     

Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect's genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the “Transposon Model” of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the “Life Cycle Model” that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.     

Exploring local consequences of two land-use alternatives for the supply of urban ecosystem services in Stockholm year 2050

Ecosystem services (ESs) are gaining ground in urban policy as a key to attaining sustainable cities. However, strategic and land-use planners need operational and accessible tools to better understand the consequences of policy and planning measures. Based on a study of the City of Stockholm and its surrounding region, we argue that spatially explicit land-use mapping is a good base for modeling and visualizing the supply of urban ESs provided by different patterns of Service Providing Units. By adding more detailed characteristics of land use through the concept of Service Providing Elements (SPEs), and by assessing synergies and trade-offs between these attributes, implications for the supply of ESs at different scale levels could be identified and discussed. Detailed land-use mapping and ES modeling were applied to two future land-use alternatives. The supply of eight urban ESs was found to vary significantly between the two alternatives depending on the ratios of different SPEs, even within identical land-use classes. One of the land-use alternatives had significantly higher potential for food and energy provision, much higher air cooling and air quality regulation capacity especially in densely built areas, showed less surface sealing, and provided better conditions for mental recreation. The exception was supply of physical recreation opportunities, where the other land-use option had an advantage. These differences became more accentuated when we zoomed in on two local urban areas. Based on these findings, our main conclusion is that, in order to provide planning and policy-making with an adequate knowledge base, it is necessary to move beyond land-use classes, as defined by European data sets like Urban Atlas, and toward tools capable of capturing more detailed aspects of land use and its relations to the supply of urban ESs. This should be made a priority, especially in early stages of planning and policy formation, and also used to support development of urban by-laws, procurement arrangements, neighborhood and building certification, etc. The approaches used in the study can serve as a valid starting point for further development of such tools and methods compatible with planners’ ordinary working modes. However, to make such progress possible, the ecosystem service research community needs to step up to the challenge of delivering locally specific and useful data on how urban land-use links to ES supply, including synergies and trade-offs between different ESs.     

短散在元件（SINE）的研究进展

短散在元件（SINE）广布于真核生物,是基因组中的可转移成分,长约100～500 bp,拷贝数可达数百至数十万以上,根据序列变异和鉴别位点常可分为若干家族和亚家族,对基因组的复杂化、基因的钝化、新基因的产生、尤其是基因表达的调控都具有重要意义.除了灵长类Alu和小鼠B1家族等少数来源于7SL RNA,大多为tRNA的衍生物,由tRNA同源区、tRNA无关区和富含AT区等组成.在tRNA同源区含有RNA聚合酶Ⅲ内部启动子成分.其起源和进化有“主源基因模型”、“多源基因模型”、“寄生假说（转座子模型）”和“平行转移假说”等.以鲑科鱼类、鲸类及偶蹄类、高等灵长类和蛙类为例,介绍了近年来短散在元件在分子系统发生研究以及在遗传作图和抗癌治疗上的应用.