An ethanol inducible gene switch for plants used to manipulate carbon metabolism.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Production and characterisation of highly concentrated nanosuspensions by high pressure homogenisation.

Nanosuspensions produced by high-pressure homogenisation are a solution for the formulation of poorly soluble drugs with bioavailability problems. The typical solid concentration of the nanosuspensions is 10%. However, to transfer the nanosuspensions to a dry product (e.g. granulation, tablets, pellets), a higher solid content is required to remove less water. Nanosuspensions with 20 and 30% solid content were produced, the effect of surfactant concentration assessed and their quality (size data) compared with the lower standard concentrations of 1-10% solid.

Production of aqueous shellac dispersions by high pressure homogenisation.

The use of shellac for coating purposes in pharmacy, food and nutriceutical industry generally involves the use of organic solvents. An aqueous shellac dispersion has been developed by applying high pressure homogenisation. Shellac was dissolved in alkaline solution, precipitated by HCl addition and the obtained suspension homogenised at 1500 bar applying 20 cycles. The volume diameter 50% was 2 microm, a distinct fraction of particles was in the nanometer range. This surfactant-free shellac dispersion with 20% solid content proved to be physically stable and showed good film forming properties.

Heavy metal contamination of nanosuspensions produced by high-pressure homogenisation.

High pressure homogenisation is a method for the production of nanosuspensions. In this process crystalline drug particles are pressed with high pressure through a narrow homogenisation gap. Due to the conditions in the gap it seems possible that metal erosion can occur. In this study the heavy metal (Fe) contamination of nanosuspensions produced by high pressure homogenisation was determined. Therefore nanosuspensions were analysed by atom absorption spectroscopy concerning their load of iron which is chosen as reference metal. The results show that the erosion of metal is below 1 ppm and will not cause any toxicological problems.

Platelet apheresis with Vivacell BT798 DEA, haemonetics V-50 and PCS-Plus: preparation efficiency and product quality using identical donors.

Different platelet apheresis techniques were used for preparing platelet concentrates: the continuous flow method with Vivacell BT 798 DEA, the discontinuous flow method with V-50 of Haemonetics, and their plasmapheresis device PCS-Plus. 20 of our donors underwent all three procedures. All platelet separations led to suitable platelet concentrates of 3 x 10(11) platelets within less than 90 min. However, the contaminating leukocytes reached 2 x 10(8). Due to less intense citrate reactions and a shorter donation time the donor acceptance of the continuous flow method was higher as compared with the discontinuous flow method.

Reduction of the cytosolic fructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield.

Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants. The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis. A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6-biphosphate in source leaves. This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO2. Measuring photosynthetic carbon fluxes by labelling leaf discs with 14CO2 revealed a 53-65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18-24%. The flux into the anionic and cationic fraction was not altered. Despite these changes steady-state sucrose concentrations were not effected in source leaves from transgenic plants. Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night. This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark. Accordingly, plant growth and potato tuber yield remained unaltered. From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy. Whether the same holds true for field conditions remains to be elucidated.

[Neonatal alloimmune thrombocytopenia--can cryopreserved thrombocytes help? (Case report)]. / Neonatale Alloimmunthrombozytopenie (NAT)--können kryokonservierte Thrombozyten helfen? (Falldemonstration).

We present a case report of the transfusion of cryopreserved HPA 1-negative platelets to a newborn suffering from neonatal alloimmune thrombocytopenia (NAT). A boy weighing 3450 g, born of a HPA1-negative mother with a platelet count of 9 Gpt/l after delivery, received two transfusions of cryopreserved HPA1-negative platelet concentrates. The first transfusion resulted in a platelet increment to 32 Gpt/l (CCI 11). A second transfusion was needed after a further drop of the platelet count on day 4 to 18 Gpt/l, resulting in an increment to 64 Gpt/l (CCI 36). Normal development during the first six months was obtained. In our opinion, cryopreserved HPA1-negative platelets could be helpful in the therapeutical strategy of NAT.

Improved red cell quality after erythroplasmapheresis with MCS-3P.

The cell separator MCS-3P is an apheresis system offering the flexibility to collect standardized red blood cells, plasma, and/or platelets from one donor. Two different programs were used for the red cell apheresis--RBCP (collection of one unit of red cells and two units of plasma) and RBCPS (one unit of red cells and one unit of plasma). The quality of the red cell concentrates (RCC resuspended in SAG-Mannitol) during the storage time of 42 days was measured by biochemical (ATP, 2,3-DPG, pH, free Hb, free potassium, glucose, lactose, p50, hemoglobin derivatives) and rheological (morphological index, filtration/rigidity index) parameters. The donation time with 53 donors was 20 min for 355 ml RCC-SAGM and 440 ml plasma and 7 min for 335 ml RCC-SAGM and 239 ml plasma. The donor tolerance was analogous to plateletpheresis or plasmapheresis. Twenty units of the RCC-SAGM were in-line filtered within 6 or 24 hours after donation. The results obtained for red blood cell storage are at least as good as with standard collection (free hemoglobin, free potassium, glucose, lactose, hemolysis) or better (ATP, 2,3-DPG, p50, hemoglobin derivatives, filtration/rigidity index) owing to prevention of collection lesion. All blood preparations were sterile after storage (red cells 42 days, plasma after freezing). The erythroplasmapheresis with MCS-3P can be especially recommended for application in an autologous blood program because the application of autologous blood donation in hospitals is often limited by the preconditions of component separation. The erythroplasmapheresis data with MCS-3P are encouraging for the development of a new blood collection methodology.

Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli.

Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylene glycol and chromatography over diethylaminoethylcellulose, omega-aminohexyl-agarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat.(mg protein)-1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121-1130). Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.

[Mechanisms of metabolic changes after in-line filtration of erythrocytes]. / Mechanismen der metabolischen Veränderungen nach einer In-Line-Filtration von roten Blutzellen.

BACKGROUND: The reasons for the improved RBC storage after in-line filtration are not fully understood. MATERIAL AND METHODS: Red blood cell concentrates were in-line filtered and stored for 6 weeks at 4 degrees C in SAGM. Parameter 2,3-DPG, p50, density, cell electrophoresis, pH were measured. RESULTS/CONCLUSION: The obtained results may explain the metabolic findings: Cell retention (during filtration older, rigide RBC are retained in the filter- a relative increase of younger RBC); changes in RBC membrane permeability (a changed electrophoretic mobility is attributed to bound RBC membrane proteins to the filter material); blood cell depletion (a high depletion efficiency prevents toxic influences of degenerating/disintegrating WBC and PLT); release of substances from filter and blood bag material (during passage of anticoagulated blood some compounds may be released in ppm concentrations - the density of in-line filtered RBC is higher than unfiltered RBC after filtration procedure and hypothermic storage).

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves.

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose-phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose-phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation.

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

The severe combined immunodeficient-human peripheral blood stem cell (SCID-huPBSC) mouse: a xenotransplant model for huPBSC-initiated hematopoiesis.

Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose-intensified anticancer chemotherapy and were transplanted into unconditioned "nonleaky" young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment.