Engineering production of antihypertensive peptides in plants

To date, a number of antihypertensive peptides (AHPs) have been identified. Most of these are derived from proteins present in common edible consumables, including milk, egg, and plant foods. Consumption of these foods serves as means of AHP delivery and thus contributing favorable health benefits. It is hypothesized that food crops, either over-expressing AHP precursor proteins or producing particular peptides as heterologous components, may serve as viable vehicles for production and delivery of functional foods as alternative hypertension therapies. In recent years, genetic engineering efforts have been undertaken to add value to functional foods. Pioneering approaches have been pursued in several crop plants, such as rice and soybean. In this review, a summary of current tools used for discovery of new AHPs, as well as strategies and perspectives of capitalizing on these AHPs in genetic engineering efforts will be presented and discussed. The implications of these efforts on the development of functional foods for preventing and treating hypertension are also presented.     

Recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals

In recent years there has been a dramatic increase in the application of plant biotechnology for the production of a variety of commercially valuable simple and complex biological molecules (biologics) for use in human and animal healthcare. Transgenic whole plants and plant cell culture systems have been developed that have the capacity to economically produce large-scale quantities of antibodies and antibody fragments, antigens and/or vaccine epitopes, metabolic enzymes, hormones, (neuro)peptides and a variety of biologically active complexes and secondary metabolites for direct use as therapeutic agents or diagnostic tools in the medical healthcare industry. As the products of genetically modified plants make their way from concept to commercialization the associated risks and acceptance by the public has been become a focal point. In this paper, we summarize the recent advances made in the use of transgenic plants and plant cell cultures as biological factories for the production of human therapeutics and biopharmaceuticals and discuss the long-term potential of `molecular farming' as a low-cost, efficient method for the production of biological materials with demonstrated utility to the pharmaceutical industry or medical community.     

Some considerations on the use of peptides and mRNA for therapeutic vaccination against cancer

Active immunization against existing cancer is a field that is currently in development and is associated with a number of problems. The potential use of peptides as minimal essential T-cell antigens and of mRNA as a novel form of antigen with advantages is discussed, with special consideration of practical aspects.     

Potential therapeutic value of muramyl peptides for modulating human immunologic responses

The clinical potential and the limitations of synthetic muramyl peptides have been suggested through extensive work describing their immunomodulating properties and toxicology. The intent of this paper is to offer the clinician a summary of these studies and to introduce the reader to the biological effects associated with administration of muramyl peptides.     

The use of antimicrotubule herbicides for the production of doubled haploid plants from anther-derived maize callus

Summary Four antimicrotubule herbicides, amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin, were evaluated for their ability to induce chromosome doubling in anther-derived, haploid maize callus. Effects of various herbicide treatments on the growth and regenerative capacity of callus along with the ploidy and seed set of regenerated plants were determined. Flow cytometric analysis was also used to measure changes in ploidy levels of callus cells following treatments. More than 50% of the cells were doubled in chromosome number after the haploid callus was treated with 5 or 10 M APM or 10 M pronamide for 3 days. A similar proportion of plants regenerated from the treated callus produced seed upon self-pollination. APM and pronamide did not inhibit callus growth at these concentrations and the treated callus retained a high plant regeneration capacity. Oryzalin very effectively induced chromosome doubling, but severely inhibited the growth of regenerable callus and plant regeneration. Trifluralin induced chromosome doubling in a small proportion of cells at lower concentrations (0.5 and 1 M), however, at a higher concentration (5 M) it inhibited callus growth and plant regeneration. The results indicate that APM and pronamide may be useful agents for inducing chromosome doubling of anther-derived maize haploid callus at very low concentrations.     

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.     

Specific peptides for the therapeutic targeting of oncogenes

Tumors are dependent on oncogenic proteins for their maintenance and survival. The ideal cancer therapy would include drugs that specifically target these proteins. Many such proteins function through interfaces that can be difficult to target effectively with small molecules. However, recent advances in cell-permeable peptide technology, improving cellular penetration and stability, raise the possibility that specific peptide interference of oncogenic proteins could be successfully translated to the clinic. Several active anti-tumor peptides were recently described. For example, a stable peptide inhibitor of the Hsp90 ATP-binding pocket killed a wide range of tumors in vitro and in vivo, and a peptide inhibitor of the BCL6 oncoprotein was active in B-cell lymphomas; both peptides functioned without toxicity to normal tissues.     

The preparation of cultured rabies virus and the production of antiserum for human use

In this paper we describe a methodology for the preparation of the Pasteur strain of fixed rabies virus in BHK-21 clone 13 cells and also its use for the production of antisera in horses. The methodology showed here is simple, rapid, facilitates the attainment of high protective titers, and the antisera produced are of high quality.     

The use of excised roots from in vitro culture for the determination of superoxide production in plants

The production of reactive oxygen species (ROS) in plants is a common event in metabolic and physiological processes as well as in the response to biotic and abiotic stress. In this paper we will report that root tissue from axenically grown tomato cultivars and Lycopersicon wild species can be used for the determination of superoxide production. Superoxide generation was evaluated following the treatment of root tissues with two general elicitors of the defence response: laminarin and calcium ionophore A23187. Results demonstrated that elicitor reactivity in terms of superoxide generation of the tomato cultivars and the wild species used was different. This suggested varying levels of competence for non-specific active defence. The proposed technique merges the advantages of in vitro cultures and of whole tissues and also demonstrates that root tissue is a suitable material for evaluating free radical release.     

Host-defense peptides of the skin with therapeutic potential: From hagfish to human

It is now well established that peptides that were first identified on the basis of their ability to inhibit growth of bacteria and fungi are multifunctional and so are more informatively described as host-defense peptides. In some cases, their role in protecting the organism against pathogenic microorganisms, although of importance, may be secondary. A previous article in the journal (Peptides 2014; 57:67–77) assessed the potential of peptides present in the skin secretions of frogs for development into anticancer, antiviral, immunomodulatory and antidiabetic drugs. This review aims to extend the scope of this earlier article by focusing upon therapeutic applications of host-defense peptides present in skin secretions and/or skin extracts of species belonging to other vertebrate classes (Agnatha, Elasmobranchii, Teleostei, Reptilia, and Mammalia as represented by the human) that supplement their potential role as anti-infectives for use against multidrug-resistant microorganisms.     

A novel delivery platform for therapeutic peptides

Although many peptides have therapeutic effects against diverse disease, their short half-lives in vivo hurdle their application as drug candidates. To extend the short elimination half-lives of therapeutic peptides, we developed a novel delivery platform for therapeutic peptides using an anti-hapten antibody and its corresponding hapten. We selected cotinine because it is non-toxic, has a well-studied metabolism, and is physiologically absent. We conjugated WKYMVm-NH₂, an anti-sepsis therapeutic peptide, to cotinine and showed that the conjugated peptide in complex with an anti-cotinine antibody has a significantly improved in vivo half-life while retaining its therapeutic efficacy. We suggest that this novel delivery platform for therapeutic peptides will be very useful to develop effective peptide therapeutics.     

The potential for protein production from green plants

Data on protein yields show that forage crops, particularly alfalfa, produce several times more protein per acre than do seed crops. Amino acid analyses and estimations of biological values by enzymatic hydrolysis and feeding trials indicate that protein concentrates from green plants have high nutritive value. The protein concentrates from 10 plant species had a similar amino acid composition and biological value which indicates that good protein might be obtained from many plant species. It is suggested that the use of the fibrous residue as a feed for ruminants and the use of the protein concentrate as a high protein feed or base for processing into new protein foods may make it possible for the production of protein from green plants to compete with other sources of protein. This would markedly increase protein production per acre and allow the use of new plant species in our agriculture. The need for more research on protein production from different types of green plants and on ways to harvest, concentrate and process their proteins into edible forms is discussed.     

Defensins: antimicrobial peptides for therapeutic development

A novel class of endogenous antimicrobial peptides called defensins has shown great versatility in their activity against a diverse range of microorganisms including bacteria, viruses and fungi. Their mode of action of bacterial cell lysis seems largely nonspecific and so promises to avert the development of resistance. These two features have made them an area of intense research activity and growing commercial interest. A successful multidisciplinary effort to investigate and develop novel defensins analogues has been established in Singapore that involves computer modeling, biochemistry, proteomics, chemical synthesis, molecular biology and clinical sciences.     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Hevein-like antimicrobial peptides of plants

Plant antimicrobial peptides represent one of the evolutionarily oldest innate immunity components providing the first line of host defense to pathogen attacks. This review is dedicated to a small, currently actively studied family of hevein-like peptides that can be found in various monocot and dicot plants. The review thoroughly describes all known pep- tides belonging to this family including data on their structures, functions, and antimicrobial activity. The main features allowing to assign these peptides to a separate family are given, and the specific characteristics of each peptide are described. Further, the mode of action for hevein-like peptides, their role in plant immune system, and the applications of these mol- ecules in biotechnology and medicine are considered.     

The production of human glucocerebrosidase in glyco‐engineered Nicotiana benthamiana plants

For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (NbGNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this NbGNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in NbGNTI‐RNAi plants (GC^gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC^gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI‐RNAi line, producing GC, was stable and the NbGNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.