Allosensitization in patients receiving multiple blood transfusions.

Extended antigen (C, E, K) matching decreased the incidence of alloantibody (alloAB) and autoantibody (autoAB) formation, in addition to eliminating transfusion reactions in the multiply transfused sickle cell disease patients. AlloAB formation possibly transforms the immune system into a hyperactive state leading to further and earlier alloAB and autoAB formation. However, additional CEK matching results in marked overuse of Rh-negative packed red blood cell (pRBC) units, 30 minutes' extra time of a skilled technologist, and 153 dollars extra CEK reagent cost per unit to find CEK-matched pRBCs for every transfusion for these multiply transfused patients.

Strategies for bioengineering the development and metabolism of glandular tissues in plants.

Glandular tissues in plants produce a wide variety of commercially important chemicals. We review specific model systems that can be exploited for bioengineering the development and metabolism of these specialized structures, and the economic considerations that must be satisfied to permit commercially viable bioengineering approaches to specific chemicals and that constrain the choice of production systems.

A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway.

Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.

Prospects for the bioengineering of isoprenoid biosynthesis.

Over the last decade, our understanding of isoprenoid biosynthesis has progressed to the stage where specific strategies for the bioengineering of essential oil production can be considered. This review provides a current overview of the enzymology and regulation of essential oil isoprenoid biosynthesis. The reaction mechanisms of the synthases which produce many of the basic isoprenoid skeletons are described in detail. Coverage is also provided of the regulation of isoprenoid biosynthesis, including the roles played by tissue and subcellular compartmentation, and by partitioning of intermediates between different branches of isoprenoid metabolism. This provides necessary context for rationally targeting specific enzymes of metabolic pathways for bioengineering essential oil production. Wherever possible, emphasis is placed on research specific to essential oil isoprenoid biosynthesis, although relevant work related to other isoprenoids is also considered when it can provide useful insights. Finally, building upon this understanding of essential oil isoprenoid biosynthesis, several approaches to the bioengineering of isoprenoid metabolism are considered.

Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway.

Procedures for the isolation and analysis of all 11 intermediates of the mevalonic acid pathway from acetyl-CoA through geranylgeranyl pyrophosphate were developed. Both acid-labile and base-labile metabolites are simultaneously extracted with good recoveries into 7 M urea at neutral pH and low temperature to minimize hydrolytic and degradative enzyme losses, and the extract is partially purified by adsorption and desorption in high yield from an anion-exchange membrane. With the use of internal standards, the pathway intermediates are subsequently separated and quantified by reversed-phase ion-pair HPLC with on-line radiodetection. Permeable secretory cells specialized for monoterpene biosynthesis were isolated from peppermint (Mentha x piperita) leaves and employed as a model system to test the analytical protocols by examining the incorporation of [14C]pyruvate, [14C]mevalonate, and [3H]isopentenyl pyrophosphate as precursors. This simple new method should be readily adaptable to a wide range of cell and tissue types that can be administered basic metabolic precursors, and should allow measurements of both flux and steady-state levels of the intermediates of the mevalonic acid pathway.

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.

Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.).

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.

Visualization of vacuoplasts in isolated vacuole preparations from mesophyll protoplasts of periwinkle [Catharanthus roseus (L.) G. Don].

A procedure was developed for the rapid detection of vacuoplasts in vacuole preparations isolated from mesophyll protoplasts of Catharanthus roseus (L.) G. Don (periwinkle). The procedure relies on the staining of surface carbohydrates on the plasma membrane surrounding vacuoplasts with fluorescein-labeled lectins. When isolated under conditions of constant osmotic strength, approximately 15-20% of the vacuoles isolated showed surface labeling with FITC-agglutinin from Abrus precatorius. Isolation of vacuoles after an initial osmotic shock showed much lower (<5%) surface labeling. This lower level of surface labeling correlated well with a lower level of other non-vacuolar marker enzyme activities. A thin layer of cytoplasm was visible in a small number of these stained structures, indicating that they were vacuoplasts.

Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts.

The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN(3) or 0.5 millimolar Cu(2+). In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu(2+), was insensitive to inhibition by NaN(3). The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics.