Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

Intermolecular ligation mediates efficient cotransformation in Phytophthora infestans

The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of nonreplicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of -glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, -galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli

Summary A system is described which enabled the selection of a heterologous ep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36000 daltons, which is in agreement with the calculated molecular weight of 35782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I gen/es.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

Anti-(transforming growth factor β) antibodies with predefined specificity inhibit metastasis of highly tumorigenic human xenotransplants in nu/nu mice

Monoclonal antibodies (mAb) were prepared against conjugated transforming growth factor 1 (TGF1) peptides: amino acid positions 48–60 and positions 86–101. Two antibodies, mAb 16-3G1 [anti-(48–60)] and mAb 5-2G6 [anti-(86–101)] cross-reacted with native TGF1,-2 and-3 (16-3G1) or only with native TGF1 (5-2G6). Both mAb were used to characterize TGF-mediated effects on the metastatic potential in nude mice of human carcinoma cell line SLU-1 and its metastatic subline SLU-M1. Autocrine TGF1-mediated up-regulation of cell proliferation and its suppression by anti-TGF antibodies in vitro was recorded for SLU-M1 cells whereas SLU-1 cell proliferation in vitro appeared to be refractory to anti-TGF antibodies and exogenous TGF-1. However, the potential of s.c. tumours to develop distant metastases in nude mice was about the same for both cell lines. Development of primary tumours and distant metastases could be suppressed by treatment of mice with anti-TGF antibodies. Thus we assume that the metastatic potential of tumour cells is independent of TGF-mediated growth-regulation effects in vitro. The anti-TGF-induced suppression of tumour progression and metastasis in nude mice might rather result from stimulation of the immune surveillance. TGF-mediated autocrine down-regulation of MHC-unrestricted cytotoxicity of activated human monocytes and CD56⁺ LAK cells and its reversion by anti-TGF antibodies could be readily demonstrated. In all our experimental series, the neutralizing potential of both anti-TGF antibodies, though directed against opposite sites of the TGF1 molecule, was very similar.     

Study of beta-amyloid peptide (Abeta40) insertion into phospholipid membranes using monolayer technique

-Amyloid peptide (A), a normal constituent of neuronal and non-neuronal cells, has been proved to be the major component of extracellular plaque of Alzheimer's disease (AD). The interaction of A with lipid membranes may be essential for its neurotoxicity. Our previous study revealed that membrane insertion may provide a possible pathway by which A prevents itself from aggregation and fibril formation. In the present work we studied the membrane insertion of A and the factors that affect its insertion ability using a monolayer approach. The results show that A is surface active and can insert into lipid monolayers. When a high level of cholesterol is present, A40 can insert into the phospholipid mixtures simulating physiological membrane composition. Acidic pH enhances A insertion, while the effect of ionic strength is rather complex. A insertion ability may be ultimately relative to cholesterol-rich domains in the monolayers, which indicates strong interaction between A and cholesterol.     

Towards the development of a bioartificial pancreas: Immunoisolation and NMR monitoring of mouse insulinomas

A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose. Cell availability limits the application of this approach at a medically-relevant scale. We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues. We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment. The system employed in this study consisted of mouse insulinoma TC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads. The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host. We fabricated a radiofrequency coil that can be double-tuned to¹H and³¹P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies. The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisition of¹H images and³¹P NMR spectra in reasonable time intervals. Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure. These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.