Urchin grazing of kelp gametophytes in warming oceans

Sea urchins can cause extensive damage to kelp forests, and their overgrazing can create extensive barren areas, leading to a loss of biodiversity. Barrens may persist when the recruitment of kelp, which occurs through the microscopic haploid gametophyte stage, is suppressed. However, the ecology of kelp gametophytes is poorly understood, and here we investigate if grazing by juvenile urchins on kelp gametophytes can suppress kelp recruitment and if this is exacerbated by climate change. We compared grazing of Ecklonia radiata gametophytes by two species of juvenile urchins, the tropical Tripneustes gratilla and the temperate Centrostephanus rodgersii, at winter (19°C), summer (23°C), and ocean warming (26°C) temperatures for the low-latitude range edge of E. radiata, which is vulnerable to ocean warming. We examined the rate of recovery of gametophytes following grazing and determined whether they survived and formed sporophytes after ingestion by sea urchins. Both T. gratilla and C. rodgersii grazed E. radiata gametophytes, reducing their abundance compared to no grazing controls. Surprisingly, temperature did not influence grazing rates, but gametophytes did not recover from grazing in the ocean warming (26°C) treatment. Gametophytes survived ingestion by both species of sea urchin and formed sporophytes after ingestion by T. gratilla, but not C. rodgersii. These results suggest complex grazer–gametophyte interactions, in which both negative (reduced abundance and poor recovery with warming) and positive (facilitated recruitment) effects are possible. Small grazers may play a more important role in kelp ecosystem function than previously thought and should be considered in our understanding of alternate stable states.     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

Successional dynamics of the cultivated kelp microbiome

Kelp are important primary producers that are colonized by diverse microbes that can have both positive and negative effects on their hosts. The kelp microbiome could support the burgeoning kelp cultivation sector by improving host growth, stress tolerance, and resistance to disease. Fundamental questions about the cultivated kelp microbiome still need to be addressed before microbiome-based approaches can be developed. A critical knowledge gap is how cultivated kelp microbiomes change as hosts grow, particularly following outplanting to sites that vary in abiotic conditions and microbial source pools. In this study we assessed if microbes that colonize kelp in the nursery stage persist after outplanting. We characterized microbiome succession over time on two species of kelp, Alaria marginata and Saccharina latissima, outplanted to open ocean cultivation sites in multiple geographic locations. We tested for host-species specificity of the microbiome and the effect of different abiotic conditions and microbial source pools on kelp microbiome stability during the cultivation process. We found the microbiome of kelp in the nursery is distinct from that of outplanted kelp. Few bacteria persisted on kelp following outplanting. Instead, we identified significant microbiome differences correlated with host species and microbial source pools at each cultivation site. Microbiome variation related to sampling month also indicates that seasonality in host and/or abiotic factors may influence temporal succession and microbiome turnover in cultivated kelps. This study provides a baseline understanding of microbiome dynamics during kelp cultivation and highlights research needs for applying microbiome manipulation to kelp cultivation.     

Host specificity and growth of kelp gametophytes symbiotic with filamentous red algae (Ceramiales,Rhodophyta)

Kelp gametophytes were previously observed in nature living endophytically in red algal cell walls. Here we examine the interactions of two kelp species and six red algae in culture. Gametophytes of Nereocystis luetkeana (Mertens) Postels et Ruprecht became endophytic in the cell walls of Griffithsia pacifica Kylin and Antithamnion defectum Kylin, and grew epiphytically in high abundance on G. japonica Okamura and Aglaothamnion oosumiense Itono. Alaria esculenta (Linnaeus) Greville from the Atlantic coast of Nova Scotia became endophytic in Aglaothamnion oosumiense, Antithamnion defectum, Callithamnion sp., G. japonica, G. pacifica, and Pleonosporium abysicola Gardner, all from the Pacific Ocean. Some cultures were treated with phloroglucinol before infection to thicken the cell walls. The endophytic gametophytes were smaller and grew more slowly than gametophytes epiphytic on the same host. N. luetkeana failed to become endophytic in some of the potential hosts, and this may reflect host specificity, or culture artifacts. This work improves our understanding of the process of infection of red algae by kelp gametophytes, and broadens our knowledge of host specificity in endophytic symbioses.Communicated by K. Lüning     

Production of human tissue-type plasminogen activator (htPA) using in vitro cultured transgenic pig mammary gland cells

Abstract

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.     

Bacterial diversity in relation to secondary production and succession on surfaces of the kelp Laminaria hyperborea

Kelp forests worldwide are known as hotspots for macroscopic biodiversity and primary production, yet very little is known about the biodiversity and roles of microorganisms in these ecosystems. Secondary production by heterotrophic bacteria associated to kelp is important in the food web as a link between kelp primary production and kelp forest consumers. The aim of this study was to investigate the relationship between bacterial diversity and two important processes in this ecosystem; bacterial secondary production and primary succession on kelp surfaces. To address this, kelp, Laminaria hyperborea, from southwestern Norway was sampled at different geographical locations and during an annual cycle. Pyrosequencing (454-sequencing) of amplicons of the 16S rRNA gene of bacteria was used to study bacterial diversity. Incorporation of tritiated thymidine was used as a measure of bacterial production. Our data show that bacterial diversity (richness and evenness) increases with the age of the kelp surface, which corresponds to the primary succession of its bacterial communities. Higher evenness of bacterial operational taxonomical units (OTUs) is linked to higher bacterial production. Owing to the dominance of a few abundant OTUs, kelp surface biofilm communities may be characterized as low-diversity habitats. This is the first detailed study of kelp-associated bacterial communities using high-throughput sequencing and it extends current knowledge on microbial community assembly and dynamics on living surfaces.     

Microprojectile bombardment of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophytes and rapid propagation of transgenic lines within a bubble-column bioreactor

A human acidic fibroblast growth factor gene, hafgf, was successfully transferred into Laminaria japonica (kelp) gametophytes via microprojectile bombardment using the biolistic PDS-1000/He gene gun. Following phosphinothricin screening, PCR detection and Southern blot analysis, transgenic L. japonica gametophytes were cultivated in an illuminated bubble-column bioreactor to optimize growth conditions. A maximal final dry cell density of 1,695 mg l⁻¹ was obtained in a batch culture having an initial dry cell density of 129.75 mg l⁻¹. This was achieved using an aeration rate of 1.08 l air min⁻¹ l⁻¹ culture in a medium containing 1.5 mM inorganic nitrate and 0.15 mM phosphate. In addition, the relationship between different nitrogen sources and growth of transgenic gametophytes indicated that both urea and sodium nitrate were effective nitrogen sources for cell growth, while ammonium ions inhibited growth of these gametophytes.     

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

Amyloid-beta levels are significantly reduced and spatial memory defects are rescued in a novel neuroserpin-deficient Alzheimer's disease transgenic mouse model

Amyloid-beta (Aβ) plaques are a hallmark of Alzheimer's disease. Several proteases including plasmin are thought to promote proteolytic cleavage and clearance of Aβ from brain. The activity of both plasmin and tissue plasminogen activator are reduced in Alzheimer's disease brain, while the tissue plasminogen activator inhibitor neuroserpin is up-regulated. Here, the relationship of tissue plasminogen activator and neuroserpin to Aβ levels is explored in mouse models. Aβ(1-42) peptide injected into the frontal cortex of tissue plasminogen activator knockout mice is slow to disappear compared to wildtype mice, whereas neuroserpin knockout mice show a rapid clearance of Aβ(1-42). The relationship of neuroserpin and tissue plasminogen activator to Aβ plaque formation was studied further by knocking-out neuroserpin in the human amyloid precursor protein-J20 transgenic mouse. Compared to the J20-transgenic mouse, the neuroserpin-deficient J20-transgenic mice have a dramatic reduction of Aβ peptides, fewer and smaller plaques, and more active tissue plasminogen activator associated with plaques. Furthermore, neuroserpin-deficient J20-transgenic mice have near normal performances in the Morris water maze, in contrast to the spatial memory defects seen in J20-transgenic mice. These results support the concept that neuroserpin inhibition of tissue plasminogen activator plays an important role both in the accumulation of brain amyloid plaques and loss of cognitive abilities.     

Shading by giant kelp canopy can restrict the invasiveness of Undaria pinnatifida (Laminariales,Phaeophyceae)

The spread of non-indigenous and invasive seaweeds has increased worldwide, and their potential effects on native seaweeds have raised concern. Undaria pinnatifida is considered among the most prolific non-indigenous species. This species has expanded rapidly in the Northeast Pacific, overlapping with native communities such as the iconic giant kelp forests (Macrocystis pyrifera). Canopy shading by giant kelp has been argued to be a limiting factor for the presence of U. pinnatifida in the understory, thus its invasiveness capacity. However, its physiological plasticity under light limitation remains unclear. In this work, we compared the physiology and growth of juvenile U. pinnatifida and M. pyrifera sporophytes transplanted to the understory of a giant kelp forest, to juveniles growing outside of the forest. Extreme low light availability compared to that outside (~0.2 and ~4.4 mol photon ⋅ m⁻² ⋅ d⁻¹, respectively) likely caused a “metabolic energy crisis” in U. pinnatifida, thus restricting its photoacclimation plasticity and nitrogen acquisition, ultimately reducing its growth. Despite M. pyrifera juveniles showing photoacclimatory responses (e.g., increases in photosynthetic efficiency and lower compensation irradiance, E_c), their physiological/vegetative status deteriorated similarly to U. pinnatifida, which explains the low recruitment inside the forest. Generally, our results revealed the ecophysiological basis behind the limited growth and survival of juvenile U. pinnatifida sporophytes in the understory.     

转基因植物表达药用蛋白的研究进展

基因工程技术的进步使得转基因植物广泛应用于工业、农业各个领域,尤其在医药制造领域。研究成果表明,转基因植物作为生物反应器在制备药用蛋白,如重组疫苗、重组动物抗体、细胞因子等方面较其他表达系统,如微生物及动物表达系统具有成本低、应用安全等优势,但在工业化技术方面仍存在障碍。     

Evidence for antheridogen production and its mediation of a mating system in natural populations of fern gametophytes

Gametophytes of Asplenium pimpinellifolium Fee and Lygodium heterodoxum Kze., occurring as natural populations in Veracruz, Mexico, were studied with respect to their spacing, size, and number of antheridia and archegonia. Small gametophytes, 0.3-2.2 mm in width and growing in colonies, usually had high numbers of antheridia. Gametophytes of the same size, growing at least 1 5 cm from the nearest neighbour, had few antheridia. In the colonial gametophytes there was a strong correlation between heavily antheridiate ones and their proximity to large archegoniate gametophytes. The data are the first to suggest the presence of an antheridogen system operating in nature, with concomitant opportunities for cross-fertilization. The rôle of polyploidy in storing genetic variation and the rôle of antheridogen-mediated out-crossing in releasing variation are seen as co-adaptive phenomena in the ferns.     

乳腺注射法表达人组织型纤溶酶原激活剂的研究

目的 :构建tPA乳腺定位表达载体 ,使其在牛乳汁中高效表达 ,观察目的基因表达的规律及其影响因素 ,为建立新型牛乳腺生物反应器提供理论基础。方法 :RT-PCR法克隆目的基因 ,通过酶切、连接、分离、纯化等方法构建含tPA-cDNA的乳腺定位表达载体 ;采用乳腺注射法将融合基因转入小鼠及牛的乳腺组织中。结果 :乳腺注射外源基因后 ,tPA可在小鼠和牛的乳汁中表达。结论 :乳腺注射法可使目的基因在乳腺组织中稳定地表达较长的时间 ,其表达量与显微注射法没有明显的差异 ,表明外源基因的表达不受转基因方法的影响。但tPA在牛乳汁中的表达量明显高于小鼠的表达量 ,提示不同动物的乳蛋白调控系统有一定的差异 ,可能受着不同的因素或调控系统的影响。     

Fish eggs as bioreactors: the production of bioactive luteinizing hormone in transgenic trout embryos

We demonstrated the production of goldfish luteinizing hormone (gfLH) by the use of 4-day-old rainbow trout embryos as novel bioreactors. This expression system has several advantages target proteins can be rapidly expressed at low cost, and recombinant proteins can be synthesized at low temperatures and can undergo complex post-translational modifications (PTMs). An expression vector containing gfLH cDNA was microinjected into fertilized trout eggs. After 4 days of incubation at 10°C, transgenic embryos were harvested and glycosylated recombinant gfLH was recovered, which stimulated testosterone production in testicular fragments from the goldfish. This is the first report on the successful production of bioactive recombinant gonadotropin originated from cyprinid. Further, these results demonstrate that trout-embryo bioreactors are a potentially powerful tool for the production of functional recombinant proteins.     

The use of Gracilaria tikvahiae (Gracilariales,Rhodophyta) as a model system to understand the nitrogen nutrition of cultured seaweeds

Seaweeds have physiological mechanisms to acquire, utilize, and store various forms of nitrogen in environments where nitrogen levels vary tremendously in space and time. Knowledge of the nitrogen relationships of seaweeds is required for the development of successful seaweed mariculture. For example, it would seem at first that continuous nitrogen enrichment would be desirable in such systems because maximal seaweed yields are possible only when growth is not nitrogen-limited. Yet such fertilization is wasteful and can result in yield reductions due to the enhancement of epiphyte growth. Because most seaweeds can rapidly taken up high concentrations of nitrogen, far in excess of what is required for current growth demands, enrichments are needed only when internal nitrogen concentrations fall to near the critical level (i.e., the minimal tissue concentration of nitrogen required for maximal growth). Nutrients are best applied at brief pulses of high nitrogen concentrations.Dedicated to the memory of Bud Brinkhuis, friend and colleague     

Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization

Many plant gums, such as gum arabic, contain hydroxyproline-rich glycoproteins (HRGPs), which are also abundant components of the plant cell extracellular matrix. Here we expressed in transgenic BY2 Nicotiana tabacum (tobacco) cells, a synthetic gene encoding a novel HRGP-based gum, designated gum arabic-8 or (GA)(8). (GA)(8) encoded eight repeats of the consensus polypeptide sequence of gum arabic glycoprotein (GAGP): Gly-Pro-His-Ser-Pro-Pro-Pro-Pro-Leu-Ser-Pro-Ser-Pro-Thr-Pro-Thr-Pro-Pro-Leu, in which most of the Pro residues were posttranslationally modified to hydroxyproline (Hyp). (GA)(8) was expressed as a green fluorescent protein (GFP) fusion protein targeted to the culture medium, (GA)(8)GFP. The culture of the transgenic cells in a 5-L bioreactor showed that the production of (GA)(8)GFP was cell growth-associated. The extracellular yield of (GA)(8)GFP was 116.8 mg/L after 14 days of culture and accounted for 87% of the total fusion protein expressed. (GA)(8)GFP was purified from the culture medium by a combination of hydrophobic interaction, gel permeation, and reversed phase chromatography. Biochemical characterization indicated that the amino acid composition of the (GA)(8) module, after removal of GFP by proteolysis, was virtually identical to that of predicted by the GAGP consensus sequence and that carbohydrate, which occurred as arabinogalactan polysaccharides and small oligoarabinosides O-linked through the Hyp residues, accounted for 84% of the molecules' dry weight. Functional assays showed that (GA)(8) exhibited low viscosity in aqueous solution similar to native GAGP. However, neither GFP alone nor the (GA)(8) module could emulsify orange oil. However, the fusion protein (GA)(8)GFP possessed 1.28-fold better emulsification properties than native GAGP. This work demonstrates the feasibility and potential of a synthetic gene approach to the de novo design of novel glycoprotein-based gums and emulsifiers.