Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration.

Blood transfusion is still a critical therapy in many diseases, traumatic events and war battlefields. However, blood cross-matching and storage may limit its applicability, especially in Third World countries. Moreover, haemoglobin, which in red blood cells is the key player in the oxygen transport from lung to tissues, when free in the plasma causes hypertension and renal failure. This investigation was aimed at the development of a novel haemoglobin-based oxygen carrier with low vasoactivity and renal filtration properties. Human haemoglobin was chemically conjugated with polyethylene glycol (PEG) under either aerobic or anaerobic conditions, following different chemical procedures. The resulting PEGylated haemoglobin products were characterized in terms of oxygen affinity, cooperativity, effects of protons and carbon dioxide concentration, and oxidation stability, and were transfused into rats to evaluate vasoactivity and renal filtration. A deoxyhaemoglobin, conjugated with seven PEG and seven propionyl groups, which we called Euro-PEG-Hb, did not produce profound hypertension, was 99% retained within 6 h, and exhibited oxygen binding properties and allosteric effects more similar to human haemoglobin A than the other tested PEGylated haemoglobin derivatives, thus appearing a very promising candidate as blood substitute.

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species.

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C(19)-GAs, including the bioactive GAs GA(1 )and GA(4), by 2beta-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA(1) and GA(4). Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds.

Drosophila melanogaster, a model system for comparative studies on the responses to real and simulated microgravity.

A key requirement to enhance our understanding of the response of biological organisms to different levels of gravity is the availability of experimental systems that can simulate microgravity and hypergravity in ground-based laboratories. This paper compares the results obtained from analysing gene expression profiles of Drosophila in space versus those obtained in a random position machine (RPM) and by centrifugation. The correlation found validates the use of the RPM simulation technique to establish the effects of real microgravity on biological systems. This work is being extended to investigate Drosophila development in another gravity modifying instrument, the levitation magnet.

Perceived safety of donor blood and blood substitutes for transfusion: the role of informational frame, patient groups and stress appraisals.

Patients express concerns about the safety of donated blood. Do they have similar concerns about potential 'blood substitutes' and does the way information is presented on these options alter patients' perceptions? A two (informational frame: gain or loss) by four (transfusion type: human donor blood, human haemoglobin, bovine haemoglobin or perfluorocarbon emulsion substitutes) by three (patient group: adult haemophiliac/leukaemia patients, relatives/friends of haemophiliac/leukaemia patients and controls) between-subjects design was used. There were 82 patients, 118 relatives/friends and 263 controls from the UK. Blood substitutes were perceived as being significantly less safe than donor blood. Perceptions of safety were greater when transfusion information (regardless of transfusion type or patient group) was presented as gains rather than losses. Different demographic and psychological factors predicted perceived safety (e.g. sex) and perceived risk (e.g. age and experience).

Trustworthiness of information about blood donation and transfusion in relation to knowledge and perceptions of risk: an analysis of UK stakeholder groups.

There is evidence that the public perceive blood transfusion as risky, and there is a growing literature exploring factors associated with perceived transfusion risk. One contributory factor might be the types of information different key stakeholders (e.g. general practitioners, anaesthetists, health/life style journalists and blood donors) rely on for information. This article addresses four unanswered questions about transfusion/donation risk, namely (1) do different stakeholder groups perceive different sources of information as more or less trustworthy, (2) to what extent is the perceived trustworthiness related to levels of knowledge and perceived risk, (3) is optimistic bias observed across the stakeholder groups, and (4) is knowledge related to levels of perceived risk across the stakeholder groups? A questionnaire survey was used to assess the trustworthiness of sources of information, perceived risk and knowledge. Four stakeholder groups were examined, namely (1) blood donors (n = 250), (2) general practitioners (n = 88), (3) anaesthetists (n = 143) and (4) medical journalists (n = 20). The results showed that (1) judgements of trustworthiness vary as a function of stakeholder groups, and (2) it is what people think they know rather than what they actually know that is related to judgements of trustworthiness and perceptions of risk. Judgements of trustworthiness are generally unrelated to perceptions of risk, and all stakeholder groups show optimistic bias. The implications of these findings for risk communication, specifically relating to transfusion medicine, are discussed.

Hemoglobin promotes somatic embryogenesis in peanut cultures.

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

Benefit and risk perceptions in transfusion medicine: blood and blood substitutes.

Blood transfusion is a remarkably safe, routine procedure in clinical medicine. However, little attention has focused on the perceptions of risk associated with the receipt of blood, blood products or 'blood substitutes'. It is pertinent to ask (i) what key stakeholder groups know about transfusion, (ii) how safe they perceive blood/blood products to be, (iii) how the latter information might influence their own and others' perceptions of risk linked to transfusion, and (iv) the extent to which approved blood substitutes might be preferred over autologous or donor blood. An appreciation of what stakeholders perceive to be the benefits and risks of the receipt of blood and blood substitutes will inform future transfusion strategies. To obtain such information, a programme of research has been initiated at Nottingham. Surveys have targeted key stakeholder groups, namely, UK adult blood donors and nondonors, anaesthetists, general practitioners and health care journalists. Experimental studies examining message framing and cueing have also been conducted with undergraduate students. Such research will improve misunderstandings about current issues associated with blood donation and transfusion against the backdrop of changing public trust of health care professionals and attitudes and expectations on blood safety and benefits of blood substitutes.

Ultrasound-induced physiological changes in cultured cells of Petunia hybrida.

Petunia hybrida cell suspension cultures were exposed to ultrasonic standing wave fields at 2.43 MHz for 40 min with mean sound pressures (within homogenous sound fields) varying from 0 (control) to ca. 1.1 MPa. Mean (+/- s.d.; n =6-9) cell viability was reduced to 87+/-10% at 0.6 MPa and to 59 +/- 23% at 1.1 MPa, compared to an initial control value of 92 +/- 6% (P <0.05). Mean (n = 3) cell alkaline phosphatase concentration increased linearly with sound pressure from a control value of 0.006+/-0.001 to 0.02+/-0.01 Sigma-Units microg(-1) protein at 1.1 MPa (P<0.05). Similarly, mean cell catalase activity increased from a control value of 0.020 +/- 0.003 to 0.026 +/- 0.008 arbitrary units at 1.1 MPa. In contrast, mean cellular lactate dehydrogenase concentration was unchanged. These observations indicate that cellular repair processes associated with increased alkaline phosphatase activity might be triggered by physical cell damage caused by ultrasound. The observed increase in catalase activity suggests increasing production of free radicals and other sonochemicals, which warrants further study. The absence of changes in lactate dehydrogenase indicates that there was no major damage to respiratory pathways or to overall cellular integrity.

Metabolic responses of cultured cells to oxygenated perfluorocarbon.

Protoplast-derived cells of albino Petunia hybrida cv. Comanche were used as a model, nonphotosynthetic, eukaryotic plant system to study changes in (1) the rate of oxygen consumption as measured by a Clark-type oxygen microelectrode, (2) mitochondrial membrane potential (MMP) as assessed by Rhodamine 123 fluorescence, and (3) intracellular activities of superoxide dismutases (SOD, EC 1.15.1.1) and catalases (CAT, EC 1.11.1.6), following culture for up to 14 d in aqueous nutrient medium overlaying oxygen-gassed perfluorodecalin (Flutec PP5; F2 Chemicals, Preston, UK). The mean (+/- s.e.m., n = 7) rate of oxygen consumption of Petunia cells after 24 h of culture in the presence of oxygenated PFC was 14.3 +/- 1.6 mol O2 ml(-1) min(-1), compared to 9.7 +/- 0.8 micromol O2 ml(-1) min(-1) for untreated (control) cells (P < 0.05). Similarly, the culture of cells with oxygenated PFC for 24 h resulted in a significant (P < 0.05) increase of over 50% in the mean MMP, compared to the control. Culture of protoplasts with oxygenated PFC also produced significant (P < 0.05) increases in both mean SOD and CAT activities after 3-7 d of culture, the former comparable to that reported previously for protoplasts of Salpiglossis sinuata cultured with oxygenated PFC.

Confidence in the safety of blood for transfusion: the effect of message framing.

BACKGROUND: Blood transfusion is a universally used, life-saving medical intervention. However, there are increasing concerns among patients about blood safety. This study investigates the effect of message framing, a means of presenting information, on confidence in blood transfusion safety. STUDY DESIGN AND METHODS: The same factual information regarding the safety of blood for transfusion was presented to a sample of 254 adult students (donors and nondonors) as either a gain frame (lives saved), a loss frame (lives lost), or a combined frame (a loss frame expressed in a positive context). This provided a basic two-way, between-subjects design with 1) blood donation history (donors vs. nondonors) and 2) message frame (gain, loss, and combined) functioning as the between-groups factors. It was hypothesized that participants would consider blood safer if information was presented as a gain frame. The role of stress appraisals as potential mediators of the framing effect was also explored. RESULTS: As predicted, participants receiving the gain-frame information were significantly more confident of the safety of blood for transfusion than those receiving loss-frame information or both. This was unaffected by donation history or appraisals of stress associated with transfusion. The extent to which blood was considered safe was negatively associated, independently of framing effects, with perceptions that transfusion was threatening. CONCLUSION: Information about transfusion should be conveyed to patients in a form focusing on the positive, rather than the negative, known facts about the safety of blood.

Hemoglobin-stimulated growth and antioxidant activities in cultured cotton cells.

Changes in cellular reactive oxygen scavenging enzymes were assessed in suspension-derived cells of cotton (Gossypium herbaceum) cv. Dhumad following culture with a commercial bovine hemoglobin (Hb) solution (Erythrogen) at 1:100-1:1000 (v:v). Mean (+/- SEM) fresh (f.wt.) and dry weights (d.wt.) of cells after 25 d of culture were significantly (p <.05) greater in medium supplemented with 1:750 and 1:1000 (v:v) Erythrogen, compared to controls lacking Erythrogen. For example, with 1:750 (v:v) Erythrogen, mean cell f.wt. and d.wt. were increased by 45 and 31%, respectively. Total soluble cellular protein increased by 141, 176, and 191% with Erythrogen at 1:50, 1:750, and 1:1000 (v:v), respectively. Cellular catalase and glutathione reductase activities decreased significantly (p <.05) following addition of low concentrations (1:1000 and 1:750 v:v) of Erythrogen to culture medium. However, increasing the concentration of Erythrogen to a maximum of 1:100 (v:v), caused a concomitant increase in catalase to a maximum of 62% over control. Mean total superoxide dismutase activity increased linearly with increasing Erythrogen concentration, reaching a maximum mean value over 2-fold greater than control with 1:100 (v:v) Erythrogen. A similar trend was observed in cellular H2O2 content, which reached a maximum of 98% over control with 1:250 (v:v) Erythrogen. These results demonstrate that culture of cotton cells with Hb solution causes changes in cellular oxygenation sufficient to modify cellular antioxidant status.

Haemoglobin (Erythrogen) enhanced microcallus formation from protoplasts of Indica rice (Oryza sativa L.).

The beneficial effects have been studied of supplementing culture medium with 1:100-1:500 (v:v) of a commercial haemoglobin solution (Erythrogen) on the mitotic division of cell suspension-derived protoplasts of Indica rices (Oryza sativa L.). Protoplasts were cultured in liquid medium, at densities of 1.5 x 10(6) or 2.5 x 10(6) ml(-1), on nitrocellulose membranes overlaying a semi-solidified medium layer that was supplemented with both Erythrogen and nurse cells of Lolium multiflorum. The mean final plating efficiencies (FPEs) of rice cv. BR26 protoplasts cultured with 1:200 (v:v) Erythrogen, at 1.5 x 10(6) ml(-1) (0.018+/-0.003%; n = 8) and 2.5 x 10(6) ml(-1) (0.016+/-0.002%; n = 8), were both significantly (P < 0.05) greater than controls lacking Erythrogen (0.0058+/-0.002%; n = 8 and 0.0041+/-0.001%; n = 8, respectively). Similarly, the mean FPEs of cv. Bini protoplasts cultured with 1:200 (v:v) Erythrogen at 1.5 x 10(6) ml(-1) (0.012+/-0.003%; n = 6) and 2.5 x 10(6) ml(-1) (0.017+/-0.001%; n = 6) were also significantly (P < 0.05) greater than their respective controls (0.003+/-0.001%, n = 6 and 0.002+/-0.001%, n = 6). In contrast, supplementation with 1:100 or 1:500 (v:v) Erythrogen did not lead to sustained mitotic division and microcallus formation in both rice cultivars.

Beneficial effects of Pluronic F-68 and artificial oxygen carriers on the post-thaw recovery of cryopreserved plant cells.

The storage of prokaryotic and eukaryotic cells at ultra-low temperature in liquid nitrogen (-196 degrees C) is a procedure that has assumed an increasingly important role in underpinning many aspects of biotechnology. For eukaryotic cells, the transition from a cryopreserved state to physiologically normal temperatures and oxygen tensions, induces respiratory imbalances that may stimulate the production of toxic oxygen radicals causing impaired cellular functions. Novel treatments, that focus specifically on enhancing oxygen delivery to cells, are important in maximising post-thaw recovery. Recently, several approaches have been evaluated with suspension cultured plant cells as a model, yet biotechnologically-important, totipotent eukaryotic cell system. Such treatments include non-ionic surfactants, primarily Pluronic F-68, and artificial oxygen carriers, the latter based on inert perfluorochemical liquids or chemically-modifed haemoglobin, as supplements to culture medium used during the post-thaw recovery phase of cell growth. When used either alone or in combination, such novel treatments stimulate significantly the post-thaw viability and biomass production of cultured plant cells. Many of these technologies will be exploitable in cryopreservation protocols for eukaryotic cells in general.

Pluronic F-68 enhanced shoot regeneration in a potentially novel citrus rootstock.

The effects have been studied in vitro of the non-ionic, co-polymer surfactant, Pluronic F-68, on shoot regeneration and bud induction in epicotyl and cotyledon explants of Citrus depressa, a potential alternative rootstock to C. jambhiri for commercial Citrus. Supplementation of Murashige and Skoog (1962)-based, agar-solidified shoot regeneration/bud induction (SRBI) medium with 1.0 mg l(-1) 6-benzylaminopurine and 0.5% (w/v) Pluronic F-68 significantly (P < 0.05) increased mean fresh weight by a maximum of 60%, the proportion of explants exhibiting shoot/bud regeneration by 25% and the mean number of shoots per epicotyl explant by 184%, compared to untreated controls. Similarly, 0.5% (w/v) Pluronic F-68 significantly (P < 0.05) enhanced the mean percentage bud induction (91%) and the number of buds regenerated (>4-fold) per cotyledon explant. Interestingly, the mean fresh weight gain for both explants was unaffected across the range of concentrations (0.001-0.1% w/v) of Pluronic F-68 evaluated. Regenerated plants from epicotyl explants were transferred and acclimatized to glasshouse conditions.

Current perceived risks of transfusion in the UK and relevance to the future acceptance of blood substitutes.

Data has been gathered on the perception of risk associated with blood donation and transfusion (including the use of so-called blood substitutes) by UK general practitioners (GPs), anaesthetists, healthcare journalists and blood donors of both genders. A questionnaire survey was conducted from March-July 2000 involving (i) GPs (n = 88), (ii) anaesthetists (n = 143), (iii)journalists (n = 20), and (iv) blood donors (n = 250). Respondents rated (scale of 1-7) the general risk of blood transfusion and the risk of infection associated with blood transfusion and donation. Respondents were asked through free response questions to identify the risks they most associated with blood transfusion and the infections associated with blood transfusion and donation. They were also asked to indicate their preference for their own blood, compared to donor blood or a blood substitute and to make a choice between donated blood or a blood substitute. The percentage of respondents who preferred to receive their own blood, compared to donor blood or a suitable substitute, was 73-94%. When required to choose between donor blood or a blood substitute, there were significant (P < 0.05) differences between sample groups: anaesthetists and GPs preferred to receive a blood substitute (52-59%), whereas blood donors and journalists preferred donated blood (74-93%). These findings have clear implications for the future development and implementation of modern transfusion options, including the use and acceptability of blood substitutes as alternatives to donor blood.

Effect of Erythrogen on post-thaw recovery of cryopreserved cell suspensions of indica rice (Oryza sativa L.).

This study shows that adding haemoglobin solution (Erythrogen) to post-thaw medium of Indica rice (Oryza sativa L.) cells enhances survival following cryopreservation. Haemoglobin (1:50 - 1:200 v:v) had a beneficial effect on post-thaw viability and subsequent cell growth. A key finding was that the successful recovery from cryopreservation of cell suspensions of the Indica rice cvs. BR26 and Pajam, and their re-establishment in AA2 medium, reflected a requirement for such supplementation of the post-thaw recovery medium with Erythrogen.

Induction of dwarfism in transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from pumpkin.

The gibberellin (GA) 20-oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi-dwarfs with smaller, deep-green leaves and highly pigmented stems compared to the wild-type. Such transformants flowered earlier than the wild-type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA(1) was reduced in leaves of the transformants to approximately 20% or less of that in the wild-type and to about 40% or less in stems. The concentrations of other 13-hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA(17), which accumulated in the transformants due to 13-hydroxylation of GA(25). By contrast, the concentrations of non-13-hydroxylated GAs, GA(4) and GA(34), were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20-oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild-type, reflecting the feedback control of 20-oxidase gene expression that serves as a homeostatic mechanism for GAs.

Viability of plant cell suspensions exposed to homogeneous ultrasonic fields of different energy density and wave type.

Exposure of Petunia hybrida cell suspensions to ultrasound at a frequency of 2.43 MHz in a standing wave field at an energy density of 70 Jm-3 (pressure amplitude of 0.78 MPa) decreased their mean viability to 35% after 20 min of sonication. A comparison of propagating wave and standing wave treatments at equal frequency (2.15 MHz) and energy density (8.5 Jm-3) showed, in the first case, a rapid decline in mean viability of cells (to 30% after 10 min of sonication) and, in the second case, a retaining of the initial viability (95%), respectively. Cells sonicated 4 days after subculture were more sensitive than cells sonicated 2 or 6 days after transfer to new culture medium. It was concluded that cellular viability depends primarily on the acoustic energy density, the exposure time, and the mechanical properties of the cells determined by age. As a consequence of the trapping of cells in the anti-node planes of the standing wave, propagating wave fields reduced cellular viability compared with standing wave fields at equal energy density.