Modulation of the NO/cGMP pathway reduces the vasoconstriction induced by acellular and PEGylated haemoglobin.

Activation of the NO/cGMP pathway modulates smooth muscle cells relaxation and hence vasoconstriction, a major hindrance for the use of cell-free haemoglobin (Hb) as blood substitute, despite conjugation with 5-kDa maleimide poly(ethylene)-glycol (PEG) reduces vasoconstriction in vivo. We aimed at assessing how a recently developed PEGylated-Hb (Deoxy-PEGHb) and manipulation of the NO/cGMP pathway enable modulation of vasoconstriction in isolated rat hearts. Hearts were Langendorff-perfused with oxygenated Krebs-Henseleit (15 ml/min) while monitoring the coronary pressure (CPP) after injection (1 min) of 50 nM norepinephrine followed by a 1 microM Hb or Deoxy-PEGHb bolus, without altering the flow. Deoxy-PEGHb induced less vasoconstriction than Hb. Although the presence of PEG could contribute to vasoconstriction, Deoxy-PEGHb did not contain appreciable amounts of free PEG. Whereas reducing endothelial NO release by 0.2 mM L-NAME increased vasoconstriction, abolishing NO scavenging by Hb using its cyanomet derivative almost completely blunted it. Furthermore, maintaining intracellular cyclic GMP by inhibiting phosphodiesterase-5 with 0.02 mM sildenafil enabled control of Hb-induced vasoconstriction. We conclude that, although PEG-Hb represents a possible approach to limit Hb-induced vasoconstriction, manipulating the NO/cGMP pathway may provide a powerful way to circumvent this problem.

The weak-acid preservative sorbic acid is decarboxylated and detoxified by a phenylacrylic acid decarboxylase, PadA1, in the spoilage mold Aspergillus niger.

Resistance to sorbic and cinnamic acids is mediated by a phenylacrylic acid decarboxylase (PadA1) in Aspergillus niger. A. niger DeltapadA1 mutants are unable to decarboxylate sorbic and cinnamic acids, and the MIC of sorbic acid required to inhibit spore germination was reduced by approximately 50% in DeltapadA1 mutants.

Identification of the sites of deoxyhaemoglobin PEGylation.

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further treated with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate 'blood substitute' in which human Hb (haemoglobin) is conjugated with long chains of PEG [poly(ethylene glycol)]. To identify the thiolation sites by MS, we have carried out the reaction using deoxyHb bound to inositol hexaphosphate to protect some of the residues crucial for function and NEM (N-ethylmaleimide) to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 PEG chains per tetramer, the thiolated residues were Lys7, Lys11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys60 of the a-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the b-chain. The a-amino groups of a- and b-chains were not modified and the reaction of the Cysb93 residues with NEM was minor or absent. After the modification with thiolane and NEM of up to five to eight lysine residues per tetramer, the products retained a large proportion of the properties of native Hb, such as low oxygen affinity, co-operativity, effect of the modulators and stability to autoxidation. Under identical anaerobic conditions, the conjugation of the thiolated Hb tetramer with five or six chains of the maleimido derivative of 6 kDa PEG yielded products with diminished co-operativity, Hill coefficient h=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to autoxidation. Co-operativity was apparently independent of the topological distribution of the PEGylated sites as obtained by treating partly the thiolated protein with NEM prior to PEGylation [poly(ethylene glycol)ation].

Circular dichroism spectroscopy of tertiary and quaternary conformations of human hemoglobin entrapped in wet silica gels.

The relative contributions to changes in visible and near UV circular dichroism spectra of hemoglobin of heme ligation and tertiary and quaternary conformational transitions were separated by exploiting the slowing down of structural relaxations for proteins encapsulated in wet, nanoporous silica gels. Spectral signatures, previously assumed to be characteristic of T and R quaternary states, were demonstrated to be specific to different tertiary conformations. The results support the view that ligation and allosteric effectors can modulate the structural and functional properties of hemoglobin by regulating the equilibrium between the same tertiary species within both quaternary states.

Isolation, culture, and plant regeneration from leaf protoplasts of Passiflora.

The family Passifloraceae contains many species exploited in the food, pharmaceutical, and ornamental plant industries. The routine culture of isolated protoplasts (naked cells) followed by reproducible plant regeneration, is crucial to the genetic improvement of Passiflora spp. by somatic cell technologies. Such procedures include somatic hybridization by protoplast fusion to generate novel hybrid plants, and gene introduction by transformation. Seedling leaves are a convenient source of totipotent protoplasts. The protoplast-to-plant system developed for Passiflora edulis fv. flavicarpa is summarized in this chapter. The procedure involves enzymatic degradation of leaf tissue using commercially-available Macerozyme R10, Cellulase R10, and Driselase. Isolated protoplasts are cultured in Kao and Michayluk medium, semi-solidified with agarose. The medium containing the suspended protoplasts is dispensed as droplets or thin layers and bathed in liquid medium of the same composition. Shoot regeneration involves transfer of protoplast-derived tissues to Murashige and Skoog-based medium. The protocols developed for P. edulis are applicable to other Passiflora spp. and will underpin the future biotechnological exploitation of a range of species in this important plant family.

Metabolite fingerprinting in transgenic lettuce.

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.

Plant protoplasts: status and biotechnological perspectives.

Plant protoplasts ("naked" cells) provide a unique single cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Several parameters, particularly the source tissue, culture medium, and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Importantly, novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. Nevertheless, isolated protoplasts are unique to a range of experimental procedures. In the context of plant genetic manipulation, somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of pharmaceutical products, and toxicological assessments. This review focuses upon the most recent developments in protoplast-based technologies.

Salinity tolerance and antioxidant status in cotton cultures.

This investigation focuses upon cell growth and antioxidant status in cultured cells of cotton (Gossypium herbaceum) cvs. Dhumad (salt-tolerant, TOL), H-14 (medium salt-tolerant, MED), and RAhs-2 (salt-sensitive, SEN) exposed to saline stress (50-200 mM NaCl). Mean (+/- SEM) callus fresh weight (f.wt.) and dry weight (d.wt.) gains were significantly (p <.05) greater on Murashige and Skoog (MS) [1]-based medium with 50 mM NaCl for the TOL cv. (62% and 16%, respectively) over NaCl-free controls (2020 +/- 45 and 166 +/- 4 mg, respectively); comparable differences were not observed for the MED cv. A significant (p <.05) decrease in mean f.wt. occurred with the SEN cv. exposed to 50 mM NaCl. For all cvs., there were (p <.05) reductions in mean f.wts. in medium with >or=100 mM NaCl. At 200 mM NaCl, mean f.wt. decreases were 52% (TOL), 89% (MED), and 91% (SEN), respectively. A strong correlation existed between antioxidant status and growth of cells with NaCl. Superoxide dismutase and glutathione reductase activities increased with increasing salinity in the TOL cv. to maximum values of 26.3 +/- 1.1 U mg(-1) protein and 1.05 +/- 0.01 AB(340 nm) min(-1) mg(-1) protein, respectively, at 150 mM NaCl; for the MED and SEN cvs., there were no changes in activities of these enzymes between control and salt treatments. Catalase activity decreased progressively with increasing salt concentration in all cvs. except for SEN with 100 mM NaCl, where mean catalase activity (1.75 +/- 0.04 AB(240 nm) min(-1) mg(-1) protein) was greater (p <.05) than control (1.13 +/- 0.08). Overall, cultured cotton cells provide an experimental system for investigating the role of antioxidants in salt tolerance at the cellular level.

Engineering blood: synthetic substitutes from fluorinated compounds.

Concerns about blood safety and the logistical problems associated with conventional transfusion have fuelled the search for effective alternatives (so-called blood substitutes). Such materials include hemoglobin derivatives and those based on synthetic, highly fluorinated, inert organic compounds called perfluorochemicals (PFCs). PFCs dissolve large volumes of oxygen and other gases, are unreactive in the body, and are excreted primarily as a vapor by exhalation. Liquid PFCs are immiscible with blood and other body fluids, but can be injected safely into the bloodstream as submicron emulsions. Emulsified PFCs have been evaluated in clinical trials as temporary, intravascular tissue-oxygenating fluids. One such emulsion, a commercial perflubron-based, phospholipid-stabilized formulation, is in advanced clinical trials as an alternative to transfusing donated (allogeneic) blood during surgery. Basic and clinical studies have shown that this emulsion can adequately maintain tissue oxygenation during acute blood loss with no abnormal hemodynamic changes. The use of PFC emulsions as an efficacious, short-term transfusion alternative underpins the longer term objective of producing a totally synthetic, bioengineered blood substitute.

Perceptions in transfusion medicine: a pilot field study on risk and ethics for blood and blood substitutes.

A pilot study was undertaken in the UK in February 2005 to identify the perceptions of risk, effectiveness and ethicality of different hypothetical transfusion options, including blood substitutes derived from different sources, among young adults. Forty-nine men and 92 women completed the questionnaire, aging between 18 and 25 years old (mean +/- standard deviation = 19.7 +/- 1.2 years). Twenty-three percent of respondents had donated blood, an average of 3.1 times. The study assessed the perceptions of donor blood versus 3 different types of potential "artificial blood" [i.e. "chemical" (synthetic), "grown from bacteria" (recombinant hemoglobin), or "based on cow blood" (bovine hemoglobin)] on three dimensions, namely risk, effectiveness, and ethicality, each scored on a 1 (least) to 7 (most) Likert-type scale. Donor blood was rated as significantly (P < 0.05) less risky, more effective and more ethical than any of the blood substitutes. The chemical-based blood substitute was rated second least risky, second most effective and second most ethical followed by bacteria grown substitute. The bovine-based blood substitute was rated as significantly riskier, least effective and least ethical. All the blood products differed significantly for perceived ethicality, with donor blood considered as most ethical and a blood substitute derived from bovine blood as least ethical. Judgments of risk correlated negatively with effectiveness (all transfusion options) and ethicality (all the blood substitutes). Overall, these results indicate that donor blood is currently preferred over blood substitutes in the UK and that judgments of risk about different hypothetical transfusion options are related to perceptions of effectiveness and ethicality.

Genomics and blood substitutes for 21st century Europe ("EuroBloodSubstitutes").

Blood transfusion is a medical intervention practised throughout the world. Blood is a biologically active material that can transmit diseases (e.g., HIV/AIDS and, perhaps, vCJD). People are becoming increasingly concerned about blood safety, despite improved screening and processing. Consequently, they are reluctant to donate blood or receive transfusions. Such problems can be solved by the development and incorporation into transfusion practices of so-called "blood substitutes" to replace some blood uses. The EuroBloodSubstitutes Project is funded by the European Union Framework 6 Programme to develop a technological platform for producing novel haem proteins and blood substitute components using micro-organisms (bacteria, fungi, yeast) as "cell factories." The Project will focus on bacteria (Escherichia coli), yeast (Pichia pastoris) and, longer-term, filamentous fungi (Aspergillus niger), all organisms used to synthesize commercially important products. The multi-centre Consortium consists of the Universities of (1) Nottingham (UK), (2) Essex (UK), (3) Denmark Technical (Denmark), (4) Lund (Sweden), (5) Milan (Italy), (6) Nancy (France), (7) Parma (Italy), (8) Rome "La Sapienza" (Italy), (9) Semmelweis (Hungary), together with (10) Alligator Bioscience, AB (Sweden), (11) LCC Engineering & Trading GmbH (Switzerland), (12) Scottish National Blood Transfusion Service (UK), and (13) Sanquin Bloodbank (The Netherlands). The EuroBloodSubstitutes Project will be informed by lay and professional stakeholders (e.g., clinicians, blood donors, patient groups, prescribers and policy makers). Outcomes of the Project are (1) the production of an information pack, decision aids and physician training aids, giving balanced overviews of the benefits and risks of transfusion of blood or potential substitutes, and (2) an interactive web site (http//:www.eurobloodsubstitutes.com) for information dissemination. This will improve knowledge and address misunderstandings about transfusion issues in a climate of changing patient expectations on blood safety and benefits of blood substitutes.

Public perception of the risk of HIV infection associated with blood donation: the role of contextual cues.

BACKGROUND: Previous surveys have reported that a high proportion of people believe that HIV can be contracted through donating blood. It was hypothesized that this may reflect an artifact of the survey methods used. This study was therefore designed to test the hypothesis that providing contextual cues specific to HIV would cause respondents to express an increased belief that the virus can be contracted by donating blood. STUDY DESIGN AND METHODS: A one-way, between-group design was used to test this hypothesis. Adult subjects (n=168) were randomly assigned to one of three experimental conditions, all three groups receiving information about the small risk of infection from blood transfusion. The variation across the experimental conditions was the example of infection given (i.e., Condition 1=no specific example of infection risk, Condition 2=data on the small risk of contracting hepatitis C through transfusion, Condition 3=data on the low risk of contracting HIV through transfusion). Respondents answered a single question, "Do you think you could catch HIV by giving (donating) blood in the UK?" RESULTS: Logistic regression analysis revealed that respondents in the HIV-cued condition were almost 11 times (OR=10.6) more likely to answer "Yes" to the survey question, as compared with the no-cue condition. There was no significant difference in response between the no-cue and the hepatitis C-cue conditions. CONCLUSION: Providing contextual cues relating to HIV increased the expressed belief that the virus could be contracted through donating blood. To better develop donor recruitment policies, future survey tools should minimize contextual effects.

Culture treatments for enhancing post-thaw recovery of cryopreserved suspension cells of potato cv. Desiree.

An efficient and reproducible protocol has been developed for the cryopreservation of cell suspension cultures of the potato (Solanum tuberosum L.) cv. Desiree. An evaluation was made of the effectiveness of different pre-culture and post-thaw treatments on cell growth, as measured by changes in biomass. Cell suspensions were cultured in UM medium supplemented with 0.25, 0.5, 0.625, 0.75 or 1.0 M sucrose prior to cryopreservation. Sucrose-treated cells were harvested from suspension and 0.75 ml packed cell volumes placed in 2 ml capacity polypropylene vials with 0.5 ml of chilled cryoprotectant (glycerol 46.0 g 1(-1), dimethylsulphoxide 39.0 g 1(-1), sucrose 342.0 g 1(-1) proline 5.0 g 1(-1); pH 5.8). Cells were frozen at -0.5 degrees C min(-1) from 0 to -35 degrees C, held at -35 degrees C for 35 min and stored, for 10 days, in liquid nitrogen (-196 degrees C). The most effective pre-treatment, in terms of subsequent post-thaw cell viability as assessed by fluorescein diacetate uptake or triphenyltetrazolium chloride reduction, was culture with 0.75 M sucrose. For this treatment, the mean absorbance (490 nm) following triphenyltetrazolium chloride reduction was 88% greater (p < 0.05) than control and 59% greater (p < 0.05) for thawed cells also cultured on supporting filter paper discs.

Agrobacterium-mediated transformation of Bangladeshi Indica rices.

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.

The weak acid preservative sorbic acid inhibits conidial germination and mycelial growth of Aspergillus niger through intracellular acidification.

The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 10(5)/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using (31)P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH(cyt)) by more than 1 pH unit and a depression of vacuolar pH (pH(vac)) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH(cyt). NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.

Quantification of a novel h-shaped ultrasonic resonator for separation of biomaterials under terrestrial gravity and microgravity conditions.

A novel, h-shaped ultrasonic resonator was used to separate biological particulates. The effectiveness of the resonator was demonstrated using suspensions of the cyanobacterium, Spirulina platensis. The key advantages of this approach were improved acoustic field homogeneity, flow characteristics, and overall separation efficiency (sigma = 1 - ratio of concentration in cleared phase to input), monitored using a turbidity sensor. The novel separation concept was also effective under microgravity conditions; gravitational forces influenced overall efficiency. Separation of Spirulina at cleared flow rates of 14 to 58 L/day, as assessed by remote video recording, was evaluated under both microgravity (