Expression and production of bioactive human interleukin-18 in transgenic tobacco plants

The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively. The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein. Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN- on IL-18-responsive J6-1 cells by the extracts obtained from the transformants. The expression level of hIL-18 (351 ng g^–1 tobacco tissue) obtained in the present study may be sufficient to induce responses/effects in vivo.     

Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Early research on the role of plastocyanin in photosynthesis

A review is presented of the early history of investigations into the function of the blue copper-protein plastocyanin in photosynthesis. The controversy or confusion that arose as to the function of plastoycanin in conjunction with cytochrome f and cytochrome c ₆ is discussed and investigations contributing to the establishment of the role of plastocyanin as the mobile electron carrier between the Photosystem I reaction center complex and the cytochrome b ₆/f complex are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

`Every dogma has its day'*: a personal look at carbon metabolism in photosynthetic bacteria

Dogmas are unscientific. What is perhaps the greatest biological dogma of all time, the `unity of biochemistry' is, in the main, still having its day. According to present knowledge, the exceptions to this dogma are mere details when seen in relation to the biosystem as a whole. Nevertheless the exceptions are scientifically interesting and the understanding of them has led to a better comprehension of photosynthesis and ecology. Until the discovery of ¹⁴C, photosynthetic CO₂ fixation was like a slightly opened black box. With ¹⁴C in hand scientists mapped out the path of carbon in green plant photosynthesis in the course of a few years. The impressive reductive pentose phosphate cycle was almost immediately assumed to be universal in autotrophs, including anoxygenic phototrophs, in spite of the odd observation to the contrary. A new dogma was born and held the field for about two decades. Events began to turn when green sulfur bacteria were found to contain ferredoxin-coupled ketoacid-oxidoreductases. This led to the formulation of a novel CO₂-fixing pathway, the reductive citric acid cycle, but its general acceptance required much work by many investigators. However, the ice had now been broken and after some years a third mechanism of CO₂ fixation was discovered, this time in Chloroflexus, and then a fourth in the same genus. One consequence of these discoveries is that it has become apparent that oxygen is an important factor that determines the kind of CO₂-fixing mechanism an organism uses. With the prospect of the characterization of hordes of novel bacteria forecast by molecular ecologists we can expect further distinctive CO₂ fixation mechanisms to turn up. This revised version was published online in August 2006 with corrections to the Cover Date.     

The contribution of photosynthetic pigments to the development of biochemical separation methods: 1900–1980

The role of photosynthetic pigments in the development of separation methods in biochemistry during the period 1900-1980 is described beginning with M. Tswett who introduced separation of chlorophylls and carotenoids on columns and coined the term chromatography in 1906. In Uppsala, T. Svedberg developed the ultracentrifuge in the 1920s. A. Tiselius improved electrophoresis in the 1930s and developed chromatography of proteins in the 1940s and 1950s. Others of 'The Uppsala school in separation science' include J. Porath, P. Flodin and S. Hjertén who further developed various gel chromatographic methods. Hjertén introduced free zone electrophoresis in narrow tubes, a forerunner of capillary electrophoresis. Two proteins, phycoerythrin and phycocyanin, were used as test substances in all these methodological studies. Aqueous two-phase partitioning as a separation method was introduced in 1956 by the author. In this work, chloroplast particles were used, and the method was applied for the separation and purification of intact chloroplasts, inside-out thylakoid vesicles and plasma membranes. My research was carried out in cooperation with G. Blomquist, G. Johansson, C. Larsson, B. Andersson and H.-E. Akerlund during a 20-year period, 1960-1980.     

Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element

A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H. polymorpha-derived integration sequence. The dimorphic yeast Arxula adeninivorans, which is currently being assessed for heterologous gene expression, was the main focus of the study. As in H. polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene. Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae. Based on these results the design of a 'universal' fungal vector appears to be feasible.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.