2 Protein Folding and its Implications for the Production of Recombinant Proteins

Abstract

Humans have utilised plant derived natural products as medicines for millenia. Moreover, many contemporary pharmaceuticals are also natural products or derivatives thereof. However, the full potential of these compounds remains to be exploited because often they are: complex and difficult to synthesise; found in low quantities; produced by undomesticated and sometimes rare plants; and, their synthesis is routinely influenced by weather conditions. Potentially, the in vitro culture of cells from the corresponding plant species could circumvent some of these problems but the growth of plant cells on an industrial scale is also problematic. The recent isolation and culture of cambial meristematic cells (CMCs), stem cells which ordinarily generate the plant vasculature, may now provide a key platform technology to help realise the full potential of plant natural products.     

Potential of plant cells in culture for cosmetic application

Plants and plant derived ingredients are common and of major importance in the fields of pharmacy, food and cosmetics. The cosmetic industry is a fast moving market. Products have short life-cycles and the industry has to come up with innovative products constantly. Most cosmetic products and their applications are defined by active ingredients. These active ingredients may derive from either synthetic sources or from plant sources. Beside this, no other origin like human or animal are accepted or allowed in cosmetics nor are genetically modified plant sources. The whole cosmetic research and development society is therefore desperately seeking for new innovative plant ingredients for cosmetic application. Unfortunately, new plant derived ingredients are limited because several plants of cosmetic interest are not to be used due to following facts: the plants contain toxic metabolites, the plants grow too slow and a seasonal harvesting is not possible, the concentration of plant constituents differ from harvest to harvest or the plant is endangered and not allowed to harvest. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plant derived actives. Due to all these findings, we decided to risk the step into plant cell culture derived cosmetic active ingredient production. This article describes the successful establishment of an apple suspension culture producing a high yield of biomass, cultured in disposable, middle-scale bioreactors. The use of a bioactive extract out of these cells for cosmetic application and the efficacy of this extract on mammalian stem cells is also outlined in this article. To obtain a suitable cosmetic product we used the high pressure homogenization technique to decompose the plant cells and release all the beneficial constituents while encapsulating these components at the same time in liquid Nanoparticles. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plants derived actives.     

Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli

Background  

Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.     

Plant cell and tissue culture: alternatives for metabolite production

Plant cell culture systems represent a potential renewable source of valuable medicinals, flavours, essences and colourants that cannot be produced by microbial cells or chemical syntheses. However, only a few cultures produce these compounds in commercially useful amounts. The low productivities are associated with our poor understanding of the biochemistry of these systems. Recent advances in molecular biology, enzymology, physiology and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important natural products. This review examines the sate of the art of production of medicinal plant secondary metabolites by plant cell cultures.     

Plant cell culture technology in the cosmetics and food industries: current state and future trends

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home.

     

Loss of TLR2, TLR4, and TLR5 on Langerhans cells abolishes bacterial recognition

It is unknown whether closely related epidermal dendritic cells, Langerhans cells (LCs), and dermal dendritic cells (DDCs) have unique functions. In this study, we show that human DDCs have a broad TLR expression profile, whereas human LCs have a selective impaired expression of cell surface TLR2, TLR4, and TLR5, all involved in bacterial recognition. This distinct TLR expression profile is acquired during the TGF-beta1-driven development of LCs in vitro. Consequently, and in contrast to DDCs, LCs weakly respond to bacterial TLR2, TLR4, and TLR5 ligands in terms of cytokine production and maturation, as well as to whole Gram-positive and Gram-negative bacteria, whereas their responsiveness to viral TLR ligands and viruses is fully active and comparable to DDCs. Unresponsiveness of LCs to bacteria may be a mechanism that contributes to tolerance to bacterial commensals that colonize the skin.     

Establishment and functional characterization of novel natural killer cell lines derived from a temperature-sensitive SV40 large T antigen transgenic mouse

Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.     

Cutting edge: virus selectively primes human langerhans cells for CD70 expression promoting CD8+ T cell responses

The two outermost compartments of skin are populated by different Ag-presenting dendritic cell types. Epidermal Langerhans cells (LCs) are evolutionarily adapted to the continuous presence of harmless skin commensals by the selective lack of cell surface TLRs that sense bacteria. In this article, we analyze the ability of LCs and dermal dendritic cells (DDCs) to respond to virus infection. Live virus and intracellular TLR3-agonist dsRNA commit LCs more effectively than DDCs to stimulate naive CD8(+) T cell expansion and their differentiation into effector cells. This potent CD8(+) T cell-promoting capacity of LCs is causally related to high levels of virus-induced CD70 expression but not to IL-12 production. These data suggest a remarkable specialization of LCs in the induction of pathogen class-specific adaptive immunity. Whereas LCs ignore bacteria, they are superior to DDCs to initiate effective CD70-mediated CD8(+) T cells in response to virus stimulation.     

Seeing tissue as a 'phase of matter': exploring statistical mechanics for the cell

The field of tissue engineering aims to produce living, biological constructs which possess the appropriate spatial ordering of cells and their extra cellular matrix products. The complexity of a single cell and its interactions in a large collective have made development of useful models to assist in tissue culture difficult, and consequentially most tissue culture endeavors are limited to trial and error approaches. Some cell types display a natural tendency to spontaneously self-assemble into large domains of parallel-oriented cells. In this work, we show that these cell culture systems can be studied in the context of continuous disorder-order phase transformations. We suggest that collective ordering of the cells is controlled by the amount of noise in the walk of the individual cells (directional persistence) because undifferentiated mesenchymal stem cells display a seven-times higher directional persistence than mature fibroblasts and have a 24-times larger final-oriented domain size, an observation that corresponds with collective ordering in self-propelled particle systems. The study of cell culture systems using analogies derived from statistical mechanics yields simple, practical models offering insight into how a long-range order can be obtained in tissue-engineered constructs, providing a new paradigm for managing operations with large collectives of living cells.     

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.     

LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant, Medicago truncatula. The analyses were conducted with plant isolates as well as the media. The LC/MS profiles of target natural products obtained from M. truncatula seedling roots, hairy roots, and suspension root cell cultures differed substantially. The most abundant compounds in seedlings roots were mono- and diglucuronides of isoflavones and/or flavones. This type of glycosylation was not observed in hairy roots or suspension root cell cultures. The only recognized glycoconjugates in the latter samples were glucose derivatives of isoflavones. Application of a high-resolution mass spectrometer helped evaluate the elemental composition of protonated molecules, such as [M + H]⁺. Comparison of collision-induced dissociation MS/MS spectra registered with a quadrupole time-of-flight analyzer for tissue extracts and standards allowed us to estimate the aglycone structure on the basis of the pseudo-MS³ experiment. Structures of these natural products were described according to the registered mass spectra and literature data. The analyses conducted represent an overview of flavonoids and their conjugates in different types of plant material representing the model legume, M. truncatula.     

Detection of natural products that induce aberrations of the mitotic spindle

A number of natural products which inhibit the synthesis and functioning of the mitotic spindle have been shown to be potent antitumour agents. As an aid to the detection and evaluation of these chemicals that produce aberrations of mitotic cell division we have utilized a model screening system based upon the simultaneous visualisation of both the mitotic spindle and the chromosomes.This assay allows the detection of morphological aberrations of both spindle synthesis and function produced by exposure to chemicals active upon the mitotic spindle. This assay has been evaluated for its ability to detect natural spindle damaging agents. Extracts of Catharanthus roseus, which produces the potent spindle toxins vinblastine and vincristine, derived from both leaves and tissue culture samples were assayed for their ability to induce spindle aberrations in human fibroblasts. Samples of extract which lacked natural vinblastine were spiked with pure vinblastine sulphate to estimate the sensitivity of the assay to detect low concentrations of known spindle toxins.This study indicates that spindle toxins may be identified in specific plant extracts at high levels of sensitivity thus providing an effective screening technique for the identification of potentially valuable plant materials for their use as sources of antitumour chemicals.     

Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.     

The highly unnatural fatty acid profile of cells in culture

The fatty acid profile of cells in culture are unlike those of natural cells with twice the monounsaturated (MUFA) and half the polyunsaturated fatty acids (PUFA) level (Mol%). This is not due to cell lines primarily being derived from cancers but is due to limited access to lipid and an inability to make PUFA de novo as vertebrate cells. Classic culture methods use media with 10% serum (the only exogenous source of lipid). Fetal bovine serum (FBS), the serum of choice has a low level of lipid and cholesterol compared to other sera and at 10% of media provides 2–3% of the fatty acid and cholesterol, 1% of the PUFA and 0.3% of the essential fatty acid linoleic acid (18:2n-6) available to cells in the body. Since vertebrate cell lines cannot make PUFA they synthesise MUFA, offsetting their PUFA deficit and reducing their fatty acid diversity. Stem and primary cells in culture appear to be similarly affected, with a rapid loss of their natural fatty acid compositions. The unnatural lipid composition of cells in culture has substantial implications for examining natural stems cell in culture, and for investigations of cellular mechanisms using cell lines based on the pervasive influence of fats.     

Detection and isolation of circulating melanoma cells using photoacoustic flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors(1,2,3). CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient's response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)(4,5). This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid(6,7). PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Pro-apoptotic versus anti-apoptotic properties of dietary resveratrol on tumoral and normal cardiac cells

Resveratrol is a natural dietary polyphenol found in grape skin, red wine, and various other food products. Resveratrol has proved to be an effective chemopreventive agent for different malignant tumors. It has also been shown to prevent vascular alterations such as atherosclerosis and inflammatory-associated events. In view of these observations, we investigated the anti-proliferative and pro-apoptotic activities of resveratrol on a tumoral cardiac cell line (HL-1 NB) derived from mouse tumoral atrial cardiac myocytes. These effects were compared with those found on normal neonatal mouse cardiomyocytes. HL-1 NB cells and neonatal cardiomyocytes were treated with resveratrol (5, 30, and/or 100 μM) for different times of culture (24, 48, and/or 72 h). Resveratrol effects were determined by various microscopical and flow cytometric methods. After resveratrol treatment, a strong inhibition of tumoral cardiac HL1-NB cell growth associated with a loss of cell adhesion was observed. This cell proliferation arrest was associated with an apoptotic process revealed by an increased percentage of cells with fragmented and/or condensed nuclei (characteristic of apoptotic cells) identified after staining with Hoechst 33342 and by the presence of cells in subG1. At the opposite, on normal cardiomyocytes, no cytotoxic effects of resveratrol were observed, and a protective effect of resveratrol against norepinephrine-induced apoptosis was found on normal cardiomyocytes. Altogether, the present data demonstrate that resveratrol (1) induces apoptosis of tumoral cardiac HL1-NB cells, (2) does not induce cell death on normal cardiomyocytes, and (3) prevents norepinephrine-induced apoptosis on normal cardiomyocytes.     

Eicosanoid release from human bronchial epithelial cells upon exposure to toluene diisocyanate in vitro

Epithelial injury and inflammation are involved in airway hyperresponsiveness and asthma induced by toluene diisocyanate. In that isocyanates are insoluble and highly reactive compounds, bronchial epithelial cells may represent the most important target cells of their toxic effect. We hypothesized that damage to airway epithelium by toluene diisocyanate may result in the release of metabolites of arachidonic acid, which are known to promote inflammation and to alter epithelial cell function and airway smooth muscle responsiveness. To test this hypothesis we examined eicosanoid products in the culture media of bronchial epithelial cells exposed in vitro to 8 and 18 ppb toluene diisocyanate. Epithelial cells derived from human bronchi obtained at surgery were cultured to confluency on collagen-coated microporous membranes. Those cells, which expressed differentiated characteristics of epithelial cells (they showed keratin-containing filaments and had a cobblestone appearance), were alternatively exposed to toluene diisocyanate or air for 30 min in a specially designed in vitro chamber. The production of metabolites of arachidonic acid was assessed by measuring the release of immunoreactive products into the cell medium at the end of the exposure and during a 2 hr period after exposure. This method revealed a predominant isocyanate-induced release of immunoreactive 15-hydroxyeicosatetraenoic acid. Release rate of this compound tended to be dose-related and was associated with cell damage as assessed by the release of lactate dehydrogenase in the medium.     

Avirulence proteins from haustoria-forming pathogens

A major insight that has emerged in the study of haustoria-forming plant pathogens over the last few years is that these eukaryotic biotrophs deliver suites of secreted proteins into host cells during infection. This insight has largely derived from successful efforts to identify avirulence (Avr) genes and their products from these pathogens. These Avr genes, identified from a rust and a powdery mildew fungus and three oomycete species, encode small proteins that are recognized by resistance proteins in the host plant cytoplasm, suggesting that they are transported inside plant cells during infection. These Avr proteins probably represent examples of fungal and oomycete effector proteins with important roles in subverting host cell biology during infection. In this respect, they represent a new opportunity to understand the basis of disease caused by these biotrophic pathogens. Elucidating how these pathogen proteins gain entry into plant cells and their biological function will be key questions for future research.     

A role for intra- and intercellular translocation in natural product biosynthesis

The formation and storage of plant natural products such as phenylpropanoids, terpenoids and alkaloids are dynamic and complex processes that involve multiple subcellular compartments and cell types. Evidence is emerging to show that consecutive enzymes of phenylpropanoid and flavonoid biosynthesis are organized into macromolecular complexes that can be associated with endomembranes, that monoterpenoid biosynthetic enzymes are exclusively localized to highly specialized glandular trichome secretory cells and that complex monoterpenoid indole- and morphinan alkaloids require a combination of phloem parenchyma, laticifers and epidermal cells for their synthesis and storage. Highly ordered, protein-mediated processes that involve intra- and intercellular translocation need be considered when attempting to understand how a plant can regulate the formation and accumulation of complex but well-defined natural product profiles.