Oxidative stress induced by fumonisin B1 in continuous human and rodent neural cell cultures

Fumonisin B₁ (FB₁) is a mycotoxin produced by Fusarium verticillioides, which is a common infectant of corn and other cereal grains. Of concern to human health is also a possible airborne exposure to FB₁-producing strains of F. verticillioides, which may grow in moisture-damaged buildings. In this study, we have characterized oxidative stress-related parameters induced by FB₁ in three different neural cell lines, human SH-SY5Y neuroblastoma, rat C6 glioblastoma and mouse GT1-7 hypothalamic cells. The cells were exposed to graded doses of FB₁ between 0.1 and 100 μM for 0-144 h after which the production of reactive oxygen species (ROS), lipid peroxidation, intracellular glutathione (GSH) levels and cell viability were measured. FB₁ caused a dose-dependent increase of ROS production in C6 glioblastoma and GT1-7 hypothalamic cells but was without an effect in SH-SY5Y cells. Decreased GSH levels, increased MDA-formation, indicative of lipid peroxidation and necrotic cell death were observed in all cell lines after incubation with FB₁. These findings indicate that FB₁ induces oxidative stress in human, rat and mouse neural cell cultures.     

Cadmium biosorption on Spirulina platensis biomass

Dry biomass of Spirulina platensis re-hydrated for 48 h was employed as a biosorbent in tests of cadmium(II) removal from water. Various concentrations of biomass (from 1 to 4 g l⁻¹) and metal (from 100 to 800 mg l⁻¹) were tested. Low biomass levels (X_o  2 g l⁻¹) ensured metal removal up to 98% only at Cd₀= 100 and 200 mg l⁻¹, while X_o  2.0 g l⁻¹ were needed at Cd₀ = 400 mg l⁻¹ to achieve satisfactory results. Whereas X_o = 4.0 g l⁻¹ was effective to remove up to Cd₀ = 500 mg l⁻¹, a further increase in metal concentration (Cd₀ = 600 and 800 mg l⁻¹) led to progressive worsening of the system performance. At a given biomass levels, the kinetics of the process was better at low Cd²⁺ concentrations, while, raising the adsorbent level from 1.0 to 2.0 g l⁻¹ and then to 4.0 g l⁻¹, the rate constant of biosorption increased by about one order of magnitude in both cases and the adsorption capacity of the system progressively decreased from 357 to 149 mg g⁻¹.     

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor

Because of the potential health risks of aflatoxin B₁ (AFB₁), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB₁ in buffer, corn and nut products. AFB₁-spiked foods were extracted with methanol and Cy5-anti-AFB₁ added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB₁. The resulting fluorescence signal decreased as the concentration of AFB₁ in the sample increased. The limit of detection for AFB₁ in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.     

Biological activities of synthetic analogues of Alternaria alternata toxin (AAL-toxin) and fumonisin in plant and mammalian cell cultures

In a search for an analogue of AAL-toxin with high phytotoxicity and low mammalian toxicity, aminopentols [(AP₁), hexacetyl AP₁ and N-acetyl AP₁], and nine analogues (1–9), were tested for toxicity to duckweed (Lemna pausicostata), susceptible tomato (asc/asc) leaf discs, black nightshade leaf discs and mammalian cell lines, including dog kidney (MDCK), rat liver hepatoma (H4TG) and mouse fibroblasts (NIH3T3). These were compared with AAL-toxin and fumonisin B₁ (FB₁). Analogue 9 at 10 μM increased cellular leakage and chlorophyll loss from both tomato and black nightshade leaf discs. The diester 9 was the most active in the duckweed bioassay, but it was much less toxic to MDCK and H4TG cells with an IC₅₀ of 200 μM compared to 10 μM for FB₁. Analogue 9 and FB₁ showed similar low toxicities (IC₅₀ = 150 μM) to NIH3T3 cells. Among the substances tested, only analogue 9 had significant phytotoxicity and low mammalian toxicity, indicating some potential for development of safe and effective natural herbicides.     

Carbon exchange and water use efficiency of a growing, irrigated olive orchard

We measured eddy covariance fluxes of CO₂ and H₂O over a flat irrigated olive orchard during growth, in different periods from Leaf Area Index (LAI) of 0.3–1.9; measurements of soil respiration were also collected. The daily net ecosystem exchange flux (F_NEE) was practically zero at LAI around 0.4 or when the orchard intercepted 11% of the incoming daily radiation; at the end of the experiment, with LAI of 1.9 (and the fraction of intercepted daily radiation close to 0.5), F_NEE was around 10 g CO₂ m⁻² day⁻¹. The night-time ecosystem respiration (R_eco), calculated from eddy fluxes in well-mixed night conditions, show a clear but non-linear dependence with LAI; it ranged from 0.05 to 0.15 mg CO₂ m⁻² s⁻¹ (in average), being the lower limit ideally close to the heterotrophic soil respiration at the site. The gross primary production flux (F_GPP) was linearly related to LAI within the LAI range of this experiment (with 11 g CO₂ m⁻² day⁻¹ increments per unit of LAI) and to the fraction of intercepted radiation. The maximum rates of F_GPP (0.75 mg CO₂ m⁻² s⁻¹) were obtained in the summer mornings of 2002, at LAI close to 1.9. F_GPP was strongly modulated by vapour pressure deficit (VPD) through the canopy conductance, even in absence of water stress. Hence, especially in the summer, the maximum rates of carbon assimilation are reached always before noon. The daily course of F_GPP shows a two-phase pattern, first related to irradiance and then to canopy conductance. The water use efficiency (WUE) was, in average, 3.8, 6.3 and 7 g CO₂ L⁻¹ in 1999, 2001 and 2002, respectively, with maxima always in the early morning. Hourly WUE was strongly related to VPD (WUE = −10.25 + 22.52 × VPD^−0.34). Our results suggest that drip irrigated orchards in general, and olive in particular, deserve specific carbon exchange and carbon budget studies and cannot be easily included in other biomes.     

基于纳米金和辣根过氧化物酶双标记抗体的黄曲霉毒素B1高灵敏检测方法的建立及应用

黄曲霉毒素B₁（aflatoxin B₁,AFB₁）在自然界普遍存在,可污染多种粮食作物和饲料,给动物和人类健康造成严重威胁。为建立AFB₁高灵敏度的快速检测方法,本研究通过采用纳米金颗粒（Au nanoparticles,AuNPs）和辣根过氧化物酶（horseradish peroxidase,HRP）双标记AFB₁单克隆抗体,建立新型酶联免疫检测方法（HRP-AuNPs IC-ELISA）,检测下限（IC₁₀）为0.017ng/mL,检测区间（IC₂₀-IC₈₀）为0.026-0.376ng/mL,半数抑制率（IC₅₀）为0.099ng/mL,与黄曲霉毒素B₂、G₁、G₂ 和M₁ 的交叉反应率分别为2.7%、9.3%、2.1%和5.3%,与赭曲霉毒素A、伏马毒素B₁、桔青霉素、展青霉毒素和玉米赤霉烯酮几乎不存在交叉反应。在玉米和面粉样本中的加标回收率可达88.93-103.55%,与LC-MS/MS同时对天然样本中AFB₁ 含量进行检测,结果表明,两种方法相关性良好。本研究建立的HRP-AuNPs IC-ELISA耗时短且灵敏度高,可用于实际样本中AFB₁ 的快速定量检测与分析,也为其他霉菌毒素的精准检测技术开发提供参考。     

The determination of fumonisin B1 in human faeces: a short term marker for assessment of exposure

Fumonisin B₁ (FB₁) is a compound that occurs frequently in rural foods and feeds, creating health hazards. When ingested, FB₁ does not appear to change in structure and is mostly excreted unchanged in faeces within 24 h. Twenty human stool samples obtained from rural school children of Vulamehlo, south of Durban (South Africa), were analysed for FB₁, as well as 23 urban control samples obtained from various households within the Durban metropolitan area. The samples were freeze-dried and ground to a fine powder. A fraction of each sample was extracted three times with aqueous ethylenediaminetetraacetic acid at pH 5.2. The pooled extracts were purified using reversed phase C₁₈ solid phase extraction cartridges. Analytical high performance liquid chromatography was used to quantitate the amount of FB₁ as an o-phthaldialdehyde (OPA) derivative in the extracts. The rural (35%) and the urban samples (9%) showed the presence of FB ranging from 790 to 19 560 ng g^-1 of freeze dried stool. It was concluded that this method could be used as a routine biomarker for short term human exposure to FB₁ in contaminated food.     

Does zaltoprofen antagonize the bradykinin receptors?

Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B₂ receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B₂ receptor antagonists (contractility, human isolated umbilical vein) or B₁ receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg⁹-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg⁹-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kinin-stimulated phospholipase A₂ activity in HEK 293 cells expressing recombinant forms of the rabbit B₁ or B₂ receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Swimming performance, oxygen consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-ranched coho salmon

Routine oxygen consumption ( M o ₂) was 35% higher in 1 day starved and 21% higher in 4 day starved adult transgenic coho salmon Oncorhynchus kisutch relative to end of migration ocean-ranched coho salmon. Critical swimming speed ( U _crit) and M o ₂ at U _crit ( M o _2max) were significantly lower in 4 day starved transgenic coho salmon (1·25 BL s⁻¹; 8·79 mg O₂ kg⁻¹ min⁻¹) compared to ocean-ranched coho salmon (1·60 BL s⁻¹; 9·87 mg O₂ kg⁻¹ min⁻¹). Transgenic fish swam energetically less efficiently than ocean-ranched fish, as indicated by a poorer swimming economy at U _crit ( M o _2max     ). Although M o _2max was lower in transgenic coho salmon, the excess post-exercise oxygen consumption (EPOC) measured during the first 20 min of recovery was significantly larger in transgenic coho salmon (44·1 mg O₂ kg⁻¹) compared with ocean-ranched coho salmon (34·2 mg O₂ kg⁻¹), which had a faster rate of recovery.     

Effect of Salvia miltiorrhiza on aflatoxin B1-induced oxidative stress in cultured rat hepatocytes

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B₁ (AFB₁). Salvia miltiorrhiza (Sm), a herbal plant that has been used extensively in traditional Chinese medicine for treating cardiovascular and liver diseases, is believed to have some antioxidative capabilities. In this study, the protective effect of Sm against AFB₁-induced cytotoxicity was investigated in cultured primary rat hepatocytes. AFB₁-induced cytotoxicity and lipid peroxidation (LPO) were estimated by determination of lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, respectively. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). In addition, changes of intracellular glutathione (GSH) content were also studied. Results showed that Sm was able to suppress the LDH leakage induced by AFB₁ in a dose-dependent manner. A dose-dependent inhibitory effect of Sm on AFB₁-induced LPO was also found in hepatocytes treated with Sm. It was further observed that Sm produced an inhibitory effect on ROS formation caused by AFB₁. Concomitantly, the GSH content in Sm-treated groups increased substantially compared to those without Sm treatment. These findings suggest that Sm can inhibit the cytotoxicity of AFB₁ through decreasing ROS formation, inhibiting LPO and preventing GSH depletion. The major component of the aqueous extract of Sm was identified by using high performance liquid chromatography (HPLC), proton magnetic resonance (¹H-NMR) and mass spectrum (MS). Analytical results suggested that D(+)β3,4-dihydroxyphenol lactic acid (DA) is the main compound of the aqueous extract of Sm.     

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus

A transgenic mouse model, deficient in kinin B₁ receptor (B₁^−/−) was used to evaluate the role of B₂ receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B₁^−/− fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B₂ receptor, we suggest that this complex was not formed in the stomach deficient in B₁ receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B₁ and B₂ receptors, an interaction between captopril and ACE, B₁ and B₂ receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.     

Bioherbicidal effect of fumonisin B1, a phytotoxic metabolite naturally produced by Fusarium nygamai, on parasitic weeds of the genus Striga

Fusarium nygamai has been isolated and identified from diseased Striga hermonthica plants collected from sorghum fields in the Sudan. Fumonisin B₁, produced by this fungus, was isolated and purified. In in vitro experiments, the bioherbicidal potential of this mycotoxin was evaluated against S. hermonthica and S. asiatica. Different concentrations (250, 500 and 1000 μg L^-1 of water) of fumonisin B₁, applied during the conditioning phase of the Striga seeds significantly reduced the germination of both species in a range of 19.8-32.2 and 34.5-47.6%, respectively. In addition, the length of the germ tubes was reduced and the germ tubes became brownish. Fumonisin B₁ slightly delayed the emergence of Striga when 250 mg fumonisin B₁ dissolved in 1 L of water were applied into the soil per pot before sowing sorghum. By the way of contrast, fumonisin B₁ was very phytotoxic when Striga seedlings (leaves and stems) of different heights and ages were sprayed with a concentration series of 250, 500 and 1000 μg fumonisin B₁ mL^-1 until run-off, or when plants were injured with a scalpel before fumonisin B₁ was applied into the injury at concentrations of 250 and 500 μg mL^-1. One day after application, the plants showed symptoms of wilting, first at the top and then moving downwards. Leaves turned black and stems desiccated at the point of injuries. Within 4 days after the application, 55% of the plants tested were completely destroyed. In total, at the end of the experiment, 40 days after the treatment, 85% of the treated plants were killed by fumonisin B_1, regardless of the concentration applied and the application technique used.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8–50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mg L⁻¹ indolebutyric acid, 0.5 mg L⁻¹ kinetin and 30 g L⁻¹ sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g (DW) L⁻¹, 4.5 mg (g DW)⁻¹ on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.     

Effect of hormones and growth factors on in vitro development of sheep preantral follicles

The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 μm) and large (>250–400 μm) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T₄), FSH, LH, transforming growth factor alpha (TGF-), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonate-buffered tissue culture medium 199, supplemented with 50 μg ml⁻¹ gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T₄ + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage.     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^-1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^-1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^-1 hair) and FB 2 (mean 11.1 μg kg^-1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Synthesis and anticancer evaluation of certain indolo[2,3-b]quinoline derivatives

The present report describes the synthesis and anticancer evaluation of certain 11-substituted 6H-indolo[2,3-b]quinolines and their methylated derivatives. These 6H-indolo[2,3-b]quinoline derivatives 11–13 were prepared from the commercially available 1,4-dihydroxyquinoline through alkylation, chlorination, nucleophilic reaction, and ring cyclization. Depending on the ratio of 11, (MeO)₂SO₂, and K₂CO₃, alkylation occurred primarily on N-5 (1:0.8:0.8) or N-6 (1:1.5:1.5) leading to the isolation of 14a or 14b as a major product. Accordingly, major product 15a (2/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 15b (1:1:1), respectively, was obtained by alkylation of 12 while 16a (13/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 16b (1:1:1), respectively, was obtained by alkylation of 13. The in vitro anticancer assay indicated 5-methylated derivatives 14a, 15a, 16a are more cytotoxic than their respective 6-methylated counterparts 14b, 15b, 16b and 6H-indolo[2,3-b]quinoline precursors 11, 12, 13. Among them, 11-(4-methoxyanilino)-6-methyl-6H-indolo[2,3-b]quinoline (16a) was the most cytotoxic with a mean GI₅₀ value of 0.78 μM and also exhibited selective cytotoxicities for HL-60 (TB), K-562, MOLT-4, RPMI-8226, and SR with GI₅₀ values of 0.11, 0.42, 0.09, 0.14, and 0.19 μM, respectively.     

Interaction of ovokinin(2-7) with vascular bradykinin 2 receptors

Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B₂ receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B₁ receptor antagonist [des-Arg¹⁰]-HOE140 (26.3±15.5%). Intracellular Ca²⁺ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg¹⁰]-HOE140. The elevation of intracellular Ca²⁺ by ovokinin(2–7) was dependent on Ca²⁺ entry from extracellular space as it was reduced in a Ca²⁺-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca²⁺ increase by the peptide. PA phosphohydrolase and phospholipase A₂ inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B₂ bradykinin receptors.     

Characterization of the B2 receptor and activity of bradykinin analogs in SHP-77 cell line by Cytosensor microphysiometer

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of measuring the rate at which cells acidify their environment in response to ligand–receptor binding. By measuring the extracellular acidification response (ECAR) we characterized some aspects of ligand–B₂ receptor interaction in SHP-77 cell line. SHP-77 cells maximally acidified their environment within 30 s after the exposure to bradykinin (BK) or the BK agonist, B9972, with the maximum effect seen at a ligands concentration of 1 μM. Fetal bovine serum (FBS) modulated the binding of BK or B9972, showing that B9972 is a partial agonist. In addition, the binding of BK agonist or antagonist to the B₂ receptor showed different ECAR and different interaction with other intracellular and plasma membrane proteins. Our microphysiometrical results showed that two parameters, antagonist binding affinity (pD₂) and antagonist potency (pIC₅₀) are required to characterize BK antagonist activity for the B₂ receptor in the SHP-77 cell line. The previously used parameter of B₂ antagonist activity, pA₂, had high variation and poor correlation with the inhibition of SHP-77 cell growth in vitro and suppression of tumor growth when SHP-77 cells were injected to mice. Our results permit us to conclude that BK agonists and antagonists differ in their interactions with the B₂ receptor and consequently elicit different cell responses. Based on our results, we have developed a new microphysiometrical assay for analyzing the activity of BK agonists and antagonist in SHP-77 cells, which may facilitate the discovery of new potent anticancer drugs.