Cloning of Fish Enzymes and Other Fish Protein Genes

ABSTRACT:?

Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.     

Implications of climate change for agricultural productivity in the early twenty-first century

This paper reviews recent literature concerning a wide range of processes through which climate change could potentially impact global-scale agricultural productivity, and presents projections of changes in relevant meteorological, hydrological and plant physiological quantities from a climate model ensemble to illustrate key areas of uncertainty. Few global-scale assessments have been carried out, and these are limited in their ability to capture the uncertainty in climate projections, and omit potentially important aspects such as extreme events and changes in pests and diseases. There is a lack of clarity on how climate change impacts on drought are best quantified from an agricultural perspective, with different metrics giving very different impressions of future risk. The dependence of some regional agriculture on remote rainfall, snowmelt and glaciers adds to the complexity. Indirect impacts via sea-level rise, storms and diseases have not been quantified. Perhaps most seriously, there is high uncertainty in the extent to which the direct effects of CO₂ rise on plant physiology will interact with climate change in affecting productivity. At present, the aggregate impacts of climate change on global-scale agricultural productivity cannot be reliably quantified.     

Food insecurity: Prevalence,causes, and the potential of transgenic `Golden Rice'

This paper reviews a number of recent social science publications on the nature and causes of food insecurity in low and middle-income countries. The focus is on one specific element of food insecurity, vitamin A deficiency, which is widespread in developing countries and causes blindness and early death among millions of children. A new approach in the fight against vitamin A deficiency is `Golden Rice'. The paper explores the pros and cons of this recently developed transgenic rice variety, and tries to answer the question whether in this particular case, genetic modification technology provides a solution to food insecurity. It is concluded that Golden Rice is one of the options available, but not necessarily the most effective one.     

Marker-assisted selection: an approach for precision plant breeding in the twenty-first century

DNA markers have enormous potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection (MAS). The large number of quantitative trait loci (QTLs) mapping studies for diverse crops species have provided an abundance of DNA marker-trait associations. In this review, we present an overview of the advantages of MAS and its most widely used applications in plant breeding, providing examples from cereal crops. We also consider reasons why MAS has had only a small impact on plant breeding so far and suggest ways in which the potential of MAS can be realized. Finally, we discuss reasons why the greater adoption of MAS in the future is inevitable, although the extent of its use will depend on available resources, especially for orphan crops, and may be delayed in less-developed countries. Achieving a substantial impact on crop improvement by MAS represents the great challenge for agricultural scientists in the next few decades.     

Trace Metals (Cd,Co, Cr,Cu, Hg,Ni, Pb,and Zn) in Food Supplements of Marine Origin

We determined the concentrations of Cd, Co, Cr, Cu, Hg, Ni, Pb, and Zn in dietary supplements of marine origin. Four supplement categories were studied; algae, coral, krill, and shark cartilage. A direct mercury analyzer was used for Hg determinations while acid digestions and ICP-AES were used for Cr analysis and ICP-MS for the other trace metals. Algae are the supplements showing the highest concentrations of Pb, Cr, and Ni with respective means of 1.6 mg Pb/kg dry weight (d.w.), 3.2 Cr mg/kg d.w., and 8.0 mg Ni/kg d.w. Krill supplements have the highest levels of Cd, Cu, and Zn with 0.65 mg Cd/kg d.w., 63 mg Cu/kg d.w., and 50 mg Zn/kg d.w., respectively. Shark cartilage supplements show the highest levels of Hg and Co with mean concentrations of 160 μg Hg/kg d.w. and 73 ± 51 μg Co/kg d.w., respectively. No samples in our study exceeded the provisional tolerable daily intakes set by Health Canada, the joint committee of the World Health Organization/Food and Agricultural Organization, or the U.S. Environmental Protection Agency. Nevertheless, Ni and Pb in algae and Hg in shark cartilage may end up contributing to a very significant portion of the allowable daily intake—leaving little room for normal intake through food consumption and other exposure pathways.     

N-Glycoprotein biosynthesis in plants: recent developments and future trends

N-glycosylation is a major modification of proteins in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide into high-mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycans occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination. At the end of their maturation, some plant N-glycans have typical structures that differ from those found in their mammalian counterpart by the absence of sialic acid and the presence of (1,2)-xylose and (1,3)-fucose residues. Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion to form mature N-glycans. On the basis of this assembly line concept, fast progress is currently made by using N-linked glycan structures as milestones of the intracellular transport of proteins along the plant secretory pathway. Further developments of this approach will need to more precisely define the topological distribution of glycosyltransferases within a plant Golgi stack. In contrast with their acknowledged role in the targeting of lysosomal hydrolases in mammalian cells, N-glycans have no specific function in the transport of glycoproteins into the plant vacuole. However, the presence of N-glycans, regardless of their structures, is necessary for an efficient secretion of plant glycoproteins. In the biotechnology field, transgenic plants are rapidly emerging as an important system for the production of recombinant glycoproteins intended for therapeutic purposes, which is a strong motivation to speed up research in plant glycobiology. In this regard, the potential and limits of plant cells as a factory for the production of mammalian glycoproteins will be illustrated.     

Analytical biotechnology of recombinant peptides and proteins: II. A confirmation of the primary structure of fusion protein containing human proinsulin and optimization of its proteolysis by trypsin

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off ofC-peptide from proinsulin results in the formation of Arg^B31-Arg^B32-insulin. The effect of temperature on the formation of de-Thr^B30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain noN-terminal Met residue. For communication I, see [1].     

植物抗冻蛋白及抗冻性分子改良

概述了植物抗冻蛋白及其相关基因的研究现状,主要包括植物低温诱导蛋白、具有热滞活性的植物抗冻蛋白及其相关基因的分离、鉴定与表达调控,以及植物抗冻性基因工程研究动态.在此基础上,讨论了该领域研究中的主要问题、发展趋势及近期研究热点.     

The impact of mineral nutrients in food crops on global human health

Nutrient sufficiency is the basis of good health, productive lives and longevity for everyone. Nutrient availability to people is primarily determined by the output of foods produced from agricultural systems. If agricultural systems fail to provide enough food diversity and quantity to satisfy all the nutrients essential to human life, people will suffer, societies will deteriorate and national development efforts will stagnate. Importantly, plant foods provide most of the nutrients that feed the developing world. Unfortunately, as a result of population pressures, many global food systems are not currently providing enough micronutrients to assure adequate micronutrient intakes for all people. This has resulted in an increasing prevalence of micronutrient deficiencies (e.g., iron deficiency, vitamin A deficiency, and iodine deficiency disorders) that now afflicts over three billion people globally mostly among resource-poor women, infants and children in developing countries. The consequences of micronutrient malnutrition are profound and alarming for human existence. Agricultural approaches to finding sustainable solutions to this problem are urgently needed. This review presents some ways in which plant nutritionists can contribute to preventing micronutrient malnutrition in sustainable ways.     

Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion,precolumn derivatization,and fluorescence detection

A sensitive and selective HPLC method for the determination of the disaccharides of chondroitin sulfate in horse and dog plasma was validated. Chondroitin sulfate is degraded by chondroitinase ABC to three primary unsaturated disaccharides, (1) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose, (2) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and (3) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose, when treated with chondroitinase. Plasma samples (0.5 ml) were treated with 50 mU of chondroitinase ABC in 50 microl of 1 mM sodium phosphate buffer (pH 7.0) at 37 degrees C for 6 h. The samples were extracted with 25% trifluoroacetic acid in ethanol. The resultant samples were derivatized with 1% dansylhydrazine in ethanol at 40 degrees C for 3 h. The chromatographic conditions consisted of fluorescence detection (excitation at 350 nm and emission at 530 nm), mu-Bondapack NH(2) (300 x 3.9 mm), and mobile phase of acetonitrile:100 mM acetate buffer, pH 5.6 (76:24), pumped at 1.0 ml/min. The standard curves for each chondroitin disaccharide showed linearity over the selected concentration range (r > or = 0.99). The intraday percentage relative standard deviation was < or =9.5% and the interday precision was < or =6.9% or less. The relative intraday and interday error ranged from -7.3 to 6.6% for each chondroitin disaccharide in the plasma. The extraction recovery was found to be in the range of 90-96%. The validated method accurately quantitated the disaccharides of chondroitin sulfate after administration to dogs and horses.     

Recombinant production of the human aquaporins in the yeast Pichia pastoris (Invited Review)

Abstract

Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism – including humans. These proteins are vital molecules and their malfunction can lead to several severe disorders and diseases. Hence, an increased understanding of their structure, function and regulation is of the utmost importance for developing current and future drugs. Heading towards this goal, the first problem to overcome is to acquire the proteins in sufficient amounts to enable functional and structural characterization. Using a suitable host organism, large amounts of target molecules can possibly be produced, but for membrane proteins limitations are frequently encountered. In the work described here, we have produced the 13 human aquaporins (hAQPs) in one of the most successful hosts for recombinant overproduction of eukaryotic proteins; the yeast Pichia pastoris, in order to explore the underlying bottleneck to a successful membrane protein production experiment. Here we present exceptional yield of hAQP1, whereas some other hAQPs were below the threshold needed for scaled up production. In the overproduction process, we have established methods for efficient production screening as well as for accurate determination of the initial production yield. Furthermore, we have optimized the yield of low producing targets, enabling studies of proteins previously out of reach, exemplified with hAQP4 as well as the homologue PfAQP. Taken together, our results. present insight into factors directing high production of eukaryotic membrane proteins together with suggestions on ways to optimize the recombinant production in the yeast P. pastoris.     

Analytical biotechnology of recombinant peptides and proteins: I. determination of the purity, composition, and structure of human, porcine, and bovine insulins

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 fromStaphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.     

蛋白质的变化与植物抗寒性的关系研究进展

蛋白质的变化在植物抗寒生理研究中一直被广泛关注。低温胁迫期间在蛋白质含量变化的同时,还可能发生质的变化,合成新的蛋白质——低温诱导蛋白。综述了低温胁迫期间植物体内蛋白质的变化,重点阐述了抗冻蛋白、脱水蛋白和热激蛋白等3种低温诱导蛋白的特性及其与植物抗寒性的关系,并对该领域今后的研究做了展望,为进一步阐明植物抗寒的分子机制、提高植物的抗寒力提供了新的思路。     

A study on verifying the effectiveness of 4-week composite weight-loss dietary supplement ingestion on body composition and blood lipid changes in middle-aged women

Purpose

The purpose of this study was to investigate the effectiveness of a composite weight-loss dietary supplement on body composition and blood lipid changes in middle-aged women.

Methods

Thirty seven middle-aged women living in the Kyunggi area participated in this study and they were randomly divided into 2 groups (Dietary supplement ingestion group; DG, n = 20 and Placebo group; PG, n = 17). Blood draw and dual energy x-ray (DEXA) measurements were conducted to examine changes in body composition and blood lipids.

Results

There were no significant changes in weight and BMI in both groups. There was an interaction between the composite weight-loss dietary supplement intake and lean body mass in DG and there was a significant decrease in percent body fat in DG. Blood lipid changes in the study results showed that there was no significant difference in TC, TG, and LDL in both groups; however, there was a significant interaction between the composite weight-loss dietary supplement intake and HDL-C as well as an increase in the HDL-C of DG.

Conclusion

In conclusion, it seems that 4-week ingestion of the composite weight-loss dietary supplement decreased body fat, increased lean body mass, and increased HDL-C. Therefore, the composite weight-loss dietary supplement is expected to prevent obesity and induce health improvements in middle-aged women.     

Coenzyme Q10 production in plants: current status and future prospects

Coenzyme Q10 (CoQ10) or Ubiquinone10 (UQ10), an isoprenylated benzoquinone, is well-known for its role as an electron carrier in aerobic respiration. It is a sole representative of lipid soluble antioxidant that is synthesized in our body. In recent years, it has been found to be associated with a range of patho-physiological conditions and its oral administration has also reported to be of therapeutic value in a wide spectrum of chronic diseases. Additionally, as an antioxidant, it has been widely used as an ingredient in dietary supplements, neutraceuticals, and functional foods as well as in anti-aging creams. Since its limited dietary uptake and decrease in its endogenous synthesis in the body with age and under various diseases states warrants its adequate supply from an external source. To meet its growing demand for pharmaceutical, cosmetic and food industries, there is a great interest in the commercial production of CoQ10. Various synthetic and fermentation of microbial natural producers and their mutated strains have been developed for its commercial production. Although, microbial production is the major industrial source of CoQ10 but due to low yield and high production cost, other cost-effective and alternative sources need to be explored. Plants, being photosynthetic, producing high biomass and the engineering of pathways for producing CoQ10 directly in food crops will eliminate the additional step for purification and thus could be used as an ideal and cost-effective alternative to chemical synthesis and microbial production of CoQ10. A better understanding of CoQ10 biosynthetic enzymes and their regulation in model systems like E. coli and yeast has led to the use of metabolic engineering to enhance CoQ10 production not only in microbes but also in plants. The plant-based CoQ10 production has emerged as a cost-effective and environment-friendly approach capable of supplying CoQ10 in ample amounts. The current strategies, progress and constraints of CoQ10 production in plants are discussed in this review.     

Techne versus Technoscience: Divergent (and Ambiguous) Notions of Food "Quality" in the French Debate over GM Crops

In the French debate over genetically modified organisms (GMOs), actors present divergent definitions of food quality located between poles of technoscience and techne. Although scientists often define food quality in terms of technoscience, assessing food safety, small farmers often appeal to technes of production, positing GMOs as a rupture with artisanal culture. Whereas small farmers (from the union the Confédération Paysanne [CP]) deploy notions of "techne" to promote their anti-GMO campaign, they often define quality in an ambiguous way, vacillating between ideas of agricultural method (technique) or production scale. Despite this ambiguity, the CP successfully designates GMOs as la malbouffe , or "bad" food, establishing themselves as protectors of artisanal technés such as Roquefort. Finally, unlike many cultures that cast GMOs as "unnatural," the CP tends to frame GMOs as "uncultural." In the French debate, the CP posits culture against a "culturelessness" associated with technoscience and industry-driven foods such as GMOs and McDonald's.     

高山和极地植物功能生态学研究进展

由于环境条件恶劣,所以高山地区（特别是树木分布线以上区域）和极地地区通常被认为是陆地上最为极端的生境之一,但是高山和极地区域却也拥有众多极具价值的生物资源。因此自1896年以来,作为生物进化研究中的热点和难点。高山和极地植物对生境的适应机制和策略一直倍受研究者们的关注。本文以植物生态学、植物生理学、气象学等学科资料,分析了高山和北极植物的特有生活型,并认为它是一种主要的适应机制。通过对全球主要高山和极地植物生长地的局部气候特点的分析,作者肯定了植物的特化适应现象与极端环境各因素间存在的密切关系。     

Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures

A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 g l^–1 after 10 d' cultivation.     

Application of high-performance tangential flow filtration (HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in Escherichia coli

High-performance tangential flow filtration (HPTFF) is shown to successfully enable concentration, purification and formulation in a single unit operation. This is illustrated with feedstreams comprising recombinant proteins expressed in Escherichia coli (E. coli). Using positively charged cellulosic membranes of 100 kDa molecular weight cut-off and operating under a selected range of buffer pH and ionic strength at a filtrate flux of 100 L m(-2) h(-1), a 10-fold removal of E. coli host cell proteins (HCP) was obtained with an overall process yield of 98%. The HPTFF performance was shown to be robust and reproducible. In addition, the novel charged membrane was regenerated and re-used seven times without loss of selectivity or throughput. When compared with a conventional purification scheme, the proposed process results in the elimination of one chromatographic step, a 12% yield improvement and a significant reduction in purification cost of goods.