Inoculation of cyprinid herpesvirus 3 (CyHV-3) on common carp brain cells-influence of process parameters on virus yield.

Research of cyprinid herpesvirus 3 (CyHV-3) is focused on the infection mechanism and disease development in animals using genetic and immunological approaches to improve treatments and diagnostics. In contrast, only few tried to investigate the CyHV-3 replication behaviour in available cell cultures. Whereas, obtaining high virus yields by in vitro replication enables achieving of the mentioned above goals easier and more reliable. The following work presents an attempt to illuminate the KHV replication in common carp brain (CCB) cell cultures from the engineering point of view. The isolate KHV-TP30 was used testing the influence on process parameters, such as multiplicity of infection (MOI), time of infection (TOI) and time of harvest (TOH). Virus concentrations and infectivity at different time points of infection were examined using hydrolyzed probe qPCR (Gilad et al. 2004) and 50% tissue culture infectivity dose (TCID50). The data obtained show that while the amount of the virus DNA remains constant after reaching its maximum, the infectivity of the virus decreases. Thus, especially, TOH can be crucial for generating a high-quality virus stock. Applying optimized parameters improved the infectivity of the harvested virus and reached a robust titre as high as 1.9 × 108 TCID50/mL. To our knowledge, so far, there is no information in the peer-reviewed literature showing comparably high virus titres. Such virus yields not only facilitate conduction of further studies, including stability tests of the virus stock under various supplementation or disinfection trails, but also provide enough virus material to perform more detailed examinations of the infection mechanism.

Antiviral activity of exopolysaccharides from Arthrospira platensis against koi herpesvirus.

Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra- and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT-PCR that EPS, applied at a concentration of >18 µg mL-1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post-infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.

Identification of newly established Spodoptera littoralis cell lines by two DNA barcoding markers.

Cell line authentication is crucial in determining the identity of cell lines and detecting any cross-contamination. The identity of three newly established Spodoptera littoralis cell lines (Spli-C, Spli-B, and Spli-S) was confirmed by DNA fingerprinting. In this study, we used two universal primers sets to amplify two DNA fragments in different positions in the mitochondrial cytochrome C oxidase 1 gene (COI). The PCR reaction succeeded in amplifying two target DNA amplicons. The first amplicon had ~650 bp, while the second had ~410 bp. By comparing the obtained informative sequences with those in the GenBank sequence database, the results showed 100% similarity between the S. littoralis cell lines and their host. The same similarity ratio was observed between the Sf21, Tni, and Cp cell lines, which are used widely, and their hosts. The informative sequences were then used for phylogenetic analyses. In addition to the high efficiency of this technique, it showed high reproducibility in two different laboratories. DNA barcoding using the two sets of the universal primers used in this study can be a fast and a reliable method for insect cell line identification.

Application of light-emitting diodes (LEDs) in cultivation of phototrophic microalgae: current state and perspectives.

The quality and regulation of the incident light is crucial in microalgae cultivation processes. Depending on wavelength, spectrum, and intensity, growth characteristics and biochemical composition of these organisms vary. With mainly fluorescent lamps (FL) used previously for illumination, such variabilities could not be studied adequately due to their broad emission spectrum. In contrast, light-emitting diodes (LEDs) emit a very narrow wavelength band and enable flexible photobioreactor designs due to their small size. This review provides a condensed overview on the application of LEDs in microalgal cultivation processes. It summarizes the current availability and applicability of LED technologies as an illumination source for research-focused photobioreactor systems. A particular focus is the use of narrow-wavelength LEDs to address fundamental as well as applied aspects of light color on algae biomass and value-added compound formation. In this respect, the application of internal and external illumination systems is reviewed together with trends in the industrial use of LED systems to intensify algae process efficiency.

Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6) cells/mLcapsule ) in semi-permeable (molecular weight cut off <10,000 Da) sodium cellulose sulfate/poly(diallyldimethylammonium chloride) polyelectrolyte capsules has previously been shown by us [Werner et al. (2013). Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells. Biotechnol Prog 29(4): 986-993]. Herein, we demonstrate that encapsulation can be used to retain the cells in continuously operated bioreactors, which opens new possibilities for research, for example, the use of Jurkat cells in pulse response experiments under steady state conditions. Two reactor concepts are presented, a fluidized and a fixed bed reactor. A direct comparison of the growth kinetics in batch and repeated batch spinner cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6) cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

Influence of the phase effect on gradient-based and statistics-based focus measures in bright field microscopy.

Autofocusing is essential to high throughput microscopy and live cell imaging and requires reliable focus measures. Phase objects such as separated single Chinese hamster ovary cells are almost invisible at the optical focus position in bright field microscopy images. Because of the phase effect, defocused images of phase objects have more contrast. In this paper, we show that widely used focus measures exhibit an untypical behaviour for such images. In the case of homogeneous cells, that is, when most cells tend to lie in the same focal plane, both gradient-based and statistics-based focus measures tend to have a local minimum instead of a global maximum at the optical focus position. On the other hand, if images show inhomogeneous cells, gradient-based focus measures tend to yield typical focus curves, whereas statistics-based focus measures deliver curves similar to the case of homogeneous cells. These results were interpreted using the equation describing the phase effect and patch-wise analysis of the focus curves. Bioprocess engineering experts are also influenced by the phase effect. Forty-four focus positions selected by them led to the conclusion that they prefer to look at defocused images instead of those at the optical focus.

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells.

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L-lysine) (Ca-alg-PLL), and a novel calcium alginate-poly(ethylene glycol) hybrid material (Ca-alg-PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca-alg-PLL capsules, while a resazurin-based assay was used for comparison in case of the Ca-alg-PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter-assay relative standard deviations below 10%.

The many faces of platelet glycoprotein Ibalpha--thrombin interaction.

The platelet glycoprotein receptor regulates the adhesion of blood platelets to damaged blood vessel walls and the subsequent platelet aggregation. One of the subunits, platelet glycoprotein Ibalpha (GpIbalpha), binds thrombin, a serine protease with both procoagulant and anticoagulant activities. Two groups reported the crystal structures of the complex between thrombin and the N-terminal extracellular domain (leucine-rich repeat [LRR] domain) of GpIbalpha. In both these structures, GpIbalpha was reported to bind two thrombin molecules, but both the primary and secondary thrombin binding sites differed between them. We performed a detailed comparison of the two structures to look for insights that may explain the differences. Our results show that the 1:1 GpIbalpha-thrombin complex detected in solution between the crystallized proteins is likely the only strong interaction. The anionic sequence (residues 268-284) of GpIbalpha is likely responsible for the initial interaction with thrombin and the interaction with the rest of LRR domain of GpIbalpha occurs subsequently and may alternate between two or more different binding modes. Our modelling suggests the interaction between GpIbalpha and thrombin is highly pH-dependent and a small change in pH is likely to contribute to the formation of alternate binding modes. The differences in the crystal structures reported for the GpIbalpha-thrombin complex suggest a fascinating plasticity in this protein-protein interaction that may be biologically significant.

Synthesis of enantiopure (5R)-hydroxyhexane-2-one with immobilised whole cells of Lactobacillus kefiri.

Whole-cell reduction of (2,5)-hexanedione to yield highly enantiopure (5R)-hydroxyhexane-2-one (enantiomeric excess >99%) with Lactobacillus kefiri DSM 20587 was investigated. Cell immobilisation with sodium cellulose sulphate was chosen as the most suitable encapsulation matrix, giving an immobilisation yield of 40%. Despite the lowered biocatalytic activity from cell immobilisation, the bioreduction process was vastly improved with the help of reaction engineering techniques (batch to a plug flow reactor set-up). High selectivity (95%) and space-time yield (87 g L(-1) day(-1)) were achieved in the plug flow reactor. The biocatalyst remained active (68% residual activity) after 6 days of operation.

Comparative analysis of the biosorption of cadmium, lead, nickel, and zinc by algae.

Thirty strains of algae were examined for their biosorption abilities in the uptake of cadmium, lead, nickel, and zinc from aqueous solution. A wide range of adsorption capacities between the different strains of algae and between the four metals can be observed. The cyanophyceae Lyngbya taylorii exhibited high uptake capacities for the four metals. The algae showed maximum capacities according to the Langmuir Adsorption Model of 1.47 mmol lead, 0.37 mmol cadmium, 0.65 mmol nickel, and 0.49 mmol zinc per gram of dry biomass. The optimum pH for L. taylorii was between pH 3 and 7 for lead, cadmium, and zinc and between pH 4 and 7 for nickel. Studies with the algae indicated a preference for the uptake of lead over cadmium, nickel, and zinc in a four metal solution. The metal binding abilities of L. taylorii could be improved by phosphorylation of the biomass. The modified biosorbent demonstrated maximum capacities of 2.52 mmol cadmium, 3.08 mmol lead, 2.79 mmol nickel, and 2.60 mmol zinc per gram of dry biomass. Investigations with phosphated L. taylorii indicated high capacities for the four metals also at low pH. The selectivity remained quite similar to the unmodified algae.

Functional capacity evaluations in persons with spinal disorders: predicting poor outcomes on the Functional Assessment Screening Test (FAST).

This study determines how performance on the simple, low exertion Functional Assessment Screening Test (FAST) relates to performance on more extensive physical and psychological testing. One hundred eighty-eight persons with chronic back disability and 17 spine healthy volunteers underwent the FAST (three 2-min static tests [kneeling, stooping, and squatting] and two 5-min tests [repetitive stooping and repetitive twisting while standing]), the Progressive Isoinertial Lifting Evaluation (PILE), trunk extension endurance, submaximal bicycle ergometry, and psychological profiles. All FAST components were completed by 88% of spine healthy subjects, but only by 19.7% (n = 37) of the back patients. Internal consistency for overall test performance was 0.82 (alpha coefficient). Back pain noncompleters had poorer performance on the PILE and trunk extension endurance despite similar cardiovascular fitness and perceived exertion during testing. They had more dysfunctional coping mechanisms, pain avoidance, depression, and self-reported disability. Since performance on nonstrenuous testing is so poor, and psychosocial variables relate strongly to test performance, extensive Functional Capacity Evaluations may not be necessary or valid in assessing the physical performance of this population of chronic back pain patients.

Divergent roles of SHP-2 in ERK activation by leptin receptors.

The protein tyrosine phosphatase SHP-2 has been proposed to serve as a regulator of leptin signaling, but its specific roles are not fully examined. To directly investigate the role of SHP-2, we employed dominant negative strategies in transfected cells. We show that a catalytically inactive mutant of SHP-2 blocks leptin-stimulated ERK phosphorylation by the long leptin receptor, ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially inhibits ERK phosphorylation. We find similar effects of the SHP-2 mutants after examining stimulation of an ERK-dependent egr-1 promoter-construct by leptin. We also demonstrate ERK phosphorylation and egr-1 mRNA expression in the hypothalamus by leptin. Analysis of signaling by ObRb lacking intracellular tyrosine residues or by the short leptin receptor, ObRa, enabled us to conclude that two pathways are critical for ERK activation. One pathway does not require the intracellular domain of ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is required for both pathways, whereas activation of ERK via Tyr-985 of ObRb also requires tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator of ERK by leptin receptors, and both the adaptor function and the phosphatase activity of SHP-2 are critical for this regulation.

Enhancement of mass transfer using colloidal liquid aphrons: measurement of mass transfer coefficients in liquid-liquid extraction.

Interphase mass transfer of a sparingly soluble solute is often the rate-limiting step in multiphase biocatalytic processes. Colloidal liquid aphrons (CLA) provide very large interfacial areas, and thus could enhance mass transfer in such processes. The aim of this study was to characterize mass transfer properties of CLA dispersions during transfer of heptanoic acid from water to limonene. The interfacial area per unit volume (a), film mass transfer coefficient (K(L)), and volumetric mass transfer coefficient (K(L)a) values were determined in a stirred-tank reactor. These results were used, along with a literature correlation, to estimate the mass transfer coefficient of the surfactant-stabilized shell surrounding the CLA. The very large increase in a provided by the CLA was only partially offset by a slight increase in the mass transfer resistance of the shell. As a result, the range of K(L)a values obtained using CLA was about an order of magnitude greater than that obtained using a conventional dispersion. The concentration of the aqueous-phase surfactant used to form the CLA strongly affected the Sauter mean diameter of the CLA; however, the concentration of the nonpolar-phase surfactant had little effect. These results suggest that CLA have considerable potential for multiphase biocatalytic applications.

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast.

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and chf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophila melanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.

Human ventricular myocytes in vitro exhibit both early and delayed preconditioning responses to simulated ischemia.

Myocardial tissue has been demonstrated to exhibit, in response to brief periods of ischemia, both an immediate period of cytoprotection [i.e. early or "first window" preconditioning response (EPR)], and a later period of cytoprotection [i.e. delayed or "second window" preconditioning response (DPR)], when exposed to a subsequent prolonged hypoxic insult. EPR has been documented in vitro in isolated cardiac myocytes, as well as in situ in intact hearts or trabeculae, for a number of vertebrate species, including humans. However, there are no reports to date of DPR in human cardiac myocytes. To address this question, human ventricular myocytes (HVM) primary isolates were prepared from fetal ventricular muscle, grown to confluency, and studied in primary culture in serum-free medium (> 90%) ventricular myocytes as determined by immunohistochemical analysis with an anti-myosin chain antibody). Using cell viability as determined by trypan blue exclusion, an EPR response could readily be detected following 15, 30, or 60 min of simulated ischemia (SI) in a hypoxic (< 1 tau pO2) buffer containing 11 mmol/l 2-deoxyglucose, followed by a prolonged (c. 17 h) SI challenge. In addition, HVM exposed to 60 min of SI, followed after 24 h by a period of SI, also exhibited a "second window" DPR (80 +/- 10% compared to 71 +/- 11% survival, in preconditioned and non-preconditioned cultures; P < 0.05; n = 18 independent experiments). Thus, in response to short periods of SI, human ventricular myocytes in vitro exhibit both "first window" and "second window" cytoprotective responses to subsequent, prolonged ischemic stress.

Intrathecal therapy of leptomeningeal CEM T-cell lymphoma in nude rats with anti-CD7 ricin toxin A chain immunotoxin.

We have established a new xenogeneic animal model of leptomeningeal metastasis (LM) by intracisternal inoculation of human CEM T-cell lymphoma into nude rats, and used it to evaluate the anti-lymphoma efficacy of an anti-CD7 ricin A chain immunotoxin (DA7). In vitro incubation with 2 microg/ml DA7 for 72 h inhibited CEM cells by 90% in a trypan blue exclusion assay. To establish its anti-lymphoma activity, one and four days after cisternal inoculation of 10(6) CEM cells, eight animals each were treated cisternally with 10 microg DA7 in 50 microl PBS or sham-treated with 50 microl PBS. Histopathologically, all eight sham-treated and five of eight DA7 treated animals showed typical features of LM with multilayers of tumor cells along the whole subarachnoid space and the ventricular walls, as well as subependymal and diffuse parenchymal tumor cell infiltration. Three DA7 treated animals were free of tumor. Two of these animals were asymptomatic long-term survivors (> 90 days). The third tumor-free animal suddenly died on day 51. Histology revealed viral myocarditis. Median symptom-free survival was 51 days (range 29-90+ days) in DA7 treated and 34 days (range 29-87 days) in sham-treated animals (p = 0.12, log-rank test). Histologically, no signs of neurotoxicity or systemic toxicity was found. However, DA7 treated animals showed a tendency to a slower weight increase on days 6-28 after tumor cell inoculation. Our results indicate that this model is useful in studying leptomeningeal seeding and intracisternal treatment of lymphoma. The demonstrated anti-tumor effect of DA7 treatment deserves further evaluation especially regarding the application of DA7 in early stages of LM from T-cell lymphoma.

Animal model of human medulloblastoma: clinical, magnetic resonance imaging, and histopathological findings after intra-cisternal injection of MHH-MED-1 cells into nude rats.

To establish an animal model of human medulloblastoma, we have injected human MHH-MED-1 cells into the cisterna magna of nude rats. Tumors grew in 3 out of 4 animals injected with 10(6) medulloblastoma cells. Affected animals showed little or no weight gain and eventually lost weight but did not develop obvious neurological symptoms until the end of observation on day 31 after inoculation. At this time, magnetic resonance imaging (MRI) in tumor-bearing rats revealed contrast enhancement in the region of the fourth ventricle and the cisterna magna. Neuropathological examination demonstrated corresponding leptomeningeal growth in the cisterna magna invading the medulla oblongata, and tumor growth within the fourth ventricle invading the pons. The tumors basically showed the same immunostaining pattern as MHH-MED-1 cells in vitro expressing neuron-specific enolase (NSE) and vimentin, but no neurofilaments (NFs), synapthophysin, or glial fibrillary acidic protein (GFAP). No tumor grew in the fourth animal, which had a normal weight gain and no alteration on MRI. In conclusion (1) the intrathecally injected human medulloblastoma cells spread similar to medulloblastomas in patients, (2) tumor growth is readily detected by MRI, (3) the new animal model is a suitable tool for further experimental research including intrathecal therapeutic studies.

Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart.

OBJECTIVE: The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS: Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS: Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS: These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.

Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in adenosine-mediated cardioprotection in rabbits, which can now be based on the appropriate recombinant rabbit A1 and A3 receptor pharmacology.