Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.     

Synthesis of a new amphiphilic glycodendrimer with antiviral functionality

A new third generation amphiphilic glycodendrimer was synthesized from a stearylamide lysine dendrimer by condensation of the oligosaccharide moiety. By stepwise condensation and deprotection of di-boc lysine from a core of stearyl amide lysine, a third-generation stearylamide lysine dendrimer was constructed. Acetyl cellobiose and glucose units with the carboxylic acid at the end of alkyl chain attached to the reducing end of the sugar moiety was condensed with surface amino groups of the third generation lysine dendrimer, respectively, to give a new stearylamide acetylcellobiose and acetylglucose lysine dendrimers. The structural analysis was carried out using NMR, IR, and matrix-associated laser desorption/ionization time-of-flight (MALDI TOF) mass spectroscopies. After deacetylation to recover hydroxyl groups and subsequent sulfation, the third-generation sulfated cellobiose stearylamide lysine dendrimer was preliminarily found to have high anti-HIV activity at a 50% effective concentration (EC(50)) as low as 6.4μg/ml and low cytotoxicity at a 50% cytotoxic concentration (CC(50)) as high as 1000μg/ml, indicating that the dendrimer gave the enhancement of the functionality of oligosaccharides with low molecular weights. The glycodendrimer with a hydrophobic stearyl chain is immobilized on hydrophobic surfaces by hydrophobic interaction and is expected to provide a new biomedical material with the surface functionality of hydrophilic sulfated oligosaccharides.     

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3′-hydroxylase (CrtZ) and β,β-carotenoid 4,4′-ketolase (4,4′-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T₀) (98.8 %) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T₁) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2 % of total carotenoids), astaxanthin monoester (18.2 %), and the free forms of astaxanthin (10.0 %) and the other ketocarotenoids (17.5 %), which indicated that artificial ketocarotenoids corresponded to 94.9 % of total carotenoids (230 μg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8 %) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.     

Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.     

Two-step purification of mouse kidney ornithine decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

Background

The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.

Results

By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis.

Conclusion

In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.