Synthesis of 18F‐labelled biotin analogues

A one‐step ¹⁸F‐labelling strategy was used to prepare three labelled analogues of the vitamin biotin, which can be useful as tracers because of biotin's high affinity for avidin. The labelled compounds were obtained in decay‐corrected yields of up to 35% and specific radioactivity of 320 GBq/µmol. When evaluated in situ, the analogues showed good affinity for avidin: 60–75% of the radiolabelled compounds were bound to avidin within 5 minutes. The binding was site‐specific, as shown by blocking experiments with native biotin.     

Brain Delivery of Biotin Bound to a Conjugate of Neutral Avidin and Cationized Human Albumin

The delivery of pharmaceuticals through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, may be facilitated by conjugation of therapeutics to brain drug delivery vectors. Since cationized albumin has been shown to undergo absorptive-mediated transcytosis through the BBB in vivo, cationized human serum albumin (cHSA) is a potential brain drug delivery vector in humans. Conjugation of biotinylated therapeutics to brain drug delivery vectors is facilitated by the preparation of vector/ avidin conjugates. Therefore, the present studies describe the preparation of a cHSA-avidin conjugate and the delivery of ³H-biotin bound to this conjugate through the BBB in vivo in anesthetized rats. Since the cationic nature of avidin (AV) accelerates the removal of avidin-based conjugates from blood in vivo, the present studies also describe the preparation and the pharmacokinetics of ³H-biotin bound to a conjugate of cHSA and neutral avidin (NLA). The bifunctional nature of the conjugate was retained: the cHSA/ NLA conjugate contained 2.8 to 6.8 biotin binding sites per conjugate, and the BBB permeability-surface area (PS) product for ³H-biotin bound to cHSA/NLA was at least 7-fold greater than the BBB PS product for ³H-biotin bound to a conjugate of NLA and native HSA (nHSA). The systemic clearance of the cHSA conjugate was reduced 10-fold by the use of NLA as opposed to AV. The increased area under the plasma concentration curve (AUC) of the cHSA-NLA conjugate correlated with an increase in brain delivery of ³H-biotin as compared to the brain delivery achieved with the cHSA/AV conjugate. In conclusion, these studies demonstrate that cHSA serves as a brain drug delivery vector and that the use of neutral forms of avidin increases the plasma AUC of the conjugate and enhances the brain delivery of biotin.     

Effects of indomethacin on ICAM-1 expression and adhesion of T lymphocytes to chondrocytes

It is now recognized that indomethacin, a frequently prescribed non-steroidal anti-inflammatory drug, may itself exert detrimental effects on cartilage matrix metabolism. In this study, the effects of indomethacin on one aspect of chondrocyte behaviour have been investigated. Using standard indirect immunolocalization techniques, it was demonstrated that chondrocytes respond to indomethacin treatment (30 μg/ml) by membrane expression of intercellular adhesion molecule, ICAM-1. Expression appeared to be augmented by combined treatment with interleukin-1α (2 ng/ml). Adhesion of T lymphocytes to chondrocyte monolayers, as measured by⁵¹Cr labelling, was promoted by indomethacin (5-30 μg/ml) and was inhibited by monoclonal anti-ICAM-1 (40 μg/ml) and an RGD-containing peptide (200 μg/ml). These results suggest that indomethacin may modulate the processes by which immunoresponsive cells accumulate in an inflammatory environment, thus exacerbating the condition and imposing a risk during long-term therapy.     

Biotinylated methotrexate loaded erythrocytes for enhanced liver uptake. 'A study on the rat'.

To serve as liver specific delivery system, methotrexate (MTX) loaded erythrocytes were modified by attachment with N-hydroxysuccinimide ester of biotin (NHS-biotin) and in vitro macrophage uptake and in vivo studies were carried out in the rat. Surface bound biotin was quantitated indirectly using an avidin support and measuring the change in absorbance at 500 nm by the interaction with 2-(4' hydroxyazobenzene) benzoic acid (HABA). A concentration course study of biotin binding reaction showed biotin molecules bound to the erythrocytes as a function of the NHS-biotin concentration. Glycerol lysis time study revealed enhanced stability of biotinylated cells (BT) compared with nonbiotinylated drug loaded cells (NB). These surface modified erythrocytes were characterized for in vitro macrophage uptake. The macrophage uptake and phagocytic index of these modified erythrocytes were enhanced by more than two-fold compared with NB cells (P<0.05). In vivo organ localization was assessed by recording amount of drug present in different organs. The modified carrier induces substantial liver targeting as drug therapeutic index in liver was found to be 3.1. These findings support the use of carrier erythrocytes, exploiting the targeting properties imparted by biotinylation. Consequently, it can spare surgical intervention to place hepatic arterial catheters for locoregional treatment of liver neoplasms.     

Immunomodulating activities of water extract fromXanthium strumarium (II): Immunostimulating effects of the water layer after treated with chloroform

One of water and/or methanol extracts from 14 herbal drugs which were screened using murine splenocytes showed immunosuppressive activities previously. After water extract fromXanthium strumarium was treated with chloroform, 100μg/ml of water layer (XS-WC1) has very strong immunostimulating activities tested by³H-thymidine incorporation (control vs 100μg/ml, 4962 cpm vs 69515 cpm). MLR also appears to be stimulated strongly (control vs 100μg/ml, 26345 cpm vs 78688 cpm). When 100μg/ml of XS-WC1 and 0.8μg/ml of concanavalin A (ConA) were added, more³H-thymidine were incorporated significantly, compared with 0.8μg/ml of ConA only. In contrast with ConA, results from 5μg/ml of lipopolysaccharide (LPS) and 100μg/ml of XS-WC1 were not different, compared with 5μg/ml of LPS only. These results indicated that responses of XS-WC1 to B cell and T cell may be different. XS-WC1 was injected intraperitoneally (10mg/kg, 50mg/kg, 100mg/kg) for 4 days or 10 days and tested secretion of IgM or IgG by direct and indirect hemolytic plaque-forming cell assays, respectively. Numbers of hemolytic plaques for both IgM and IgG were increased significantly. Especially, secretion of IgGs was increased more than 10 times. After administration of XS-WC1 for 7 days (50mg/kg, 100mg/kg) splenomegaly due to graft vs host reaction was observed. Human lymphocytes separated from whole blood by Ficoll-Hypaque method were also proliferated after treatment of 10μg/ml and 50μg/ml of XS-WC1. As seen in murine lymphocytes, human lymphocyte proliferation was increased synergistically after treatment with both of XS-WC1 and phytohemagglutinin (PHA). It appears that XS-WC1 may have potential immunostimulating activities and that it remains to be purified further for isolation of active components.     

Novel Microneedle Patches for Active Insulin Delivery are Efficient in Maintaining Glycaemic Control: An Initial Comparison with Subcutaneous Administration

Purpose Good glycaemic control is essential to minimize the risk for diabetes-induced complications. Also, compliance is likely to be higher if the procedure is simple and painless. This study was designed to validate painless intradermal delivery via a patch-like microneedle array. Materials and Methods Diabetes was induced by an intravenous injection of streptozotocin (50 mg/kg bw) in adult male Sprague Dawley rats. Plasma insulin and blood glucose were measured before, during and after subcutaneous or intradermal (microneedles) infusion of insulin (0.2 IU/h) under Inactin-anaesthesia. Results Before insulin administration, all animals displayed a pronounced hyperglycaemia (19 ± 1 mM; 359 mg/dl). Administration of insulin resulted in a reduced plasma glucose independently of administration route (subcutaneous 7.5 ± 4.2, n = 9, and intradermal 11 ± 1.8, n = 9 after 240 min), but with less errors of the mean in the intradermal group. In the intradermal group, plasma insulin was increased in all latter measurements (72 ± 22, 81 ± 34, and 87 ± 20 μIU/ml), as compared to the first measurement (26 ± 13). In the subcutaneous group, plasma insulin was elevated during the last measurement (to 154 ± 3.5 μIU/ml from 21 ± 18). Conclusion This study presents a novel possibility of insulin delivery that is controllable and requires minimal training. This treatment strategy could improve compliance, and thus be beneficial for patients’ glycaemic control.     

Drug targeting to the brain using avidin-biotin technology in the mouse; (blood-brain barrier, monoclonal antibody, transferrin receptor, Alzheimer's disease)

A beta1-40 peptide radiopharmaceuticals could be used to image A beta brain amyloid in transgenic mouse models of Alzheimer's disease should the A beta peptide radiopharmaceutical be made transportable through the blood-brain barrier (BBB) in vivo. The present studies used the RI7-217 rat monoclonal antibody to the mouse transferrin receptor as a BBB drug targeting vector for the delivery to brain of A beta1-40 radiolabeled with either 125-Iodine or 111-Indium. The A beta peptide radiopharmaceutical is conjugated to the RI7 MAb using avidin biotin technology, wherein the A beta1-40 peptide radiopharmaceutical is monobiotinylated (bio) and bound to a conjugate of the RI7 MAb and streptavidin (SA). The [125 I]-bio-A beta1-40 or the [111 In]-bio-A beta1-40 either free or bound to the RI7/SA conjugate was injected intravenously into anesthetized adult mice and plasma pharmacokinetics and organ uptake were measured over the next 60 minutes. The A beta1-40 peptide radiopharmaceutical radiolabeled with 111-Indium was the preferred formulation, compared to peptide labeled with 125-Iodine, because there was a greater metabolic stability and reduced artifactual organ uptake of metabolites associated with the use of the 111-Indium nuclide. However, biotinylated A beta1-40 peptide radiopharmaceuticals conjugated to the RI7/SA brain drug targeting system were metabolically unstable in mice in vivo owing to active biotinidase activity. Future work involving brain drug targeting in mice that utilizes avidin biotin technology will need to incorporate biotin analogues that are resistant to biotinidase.     

Polyoxygenated flavones; Synthesis,cytotoxicities and antitumor activity against ICR mice carrying S-180 cells

Fifty two flavones were synthesized from polyoxygenated dibenzoylmethanes which were obtained by a modified Baker-Venkataraman rearrangement, of 2-benzoyl oxyacetophenones. The following flavones among then showed good cytotoxic activities against L1210 and HL-60 cells; 2′-benzyloxy-5-methoxyflavone [ED₅₀(L1210)=4.9 μg/ml) ED₅₀(HL-60)=3.1 μg/ml] 2′-benzyloxy-5,7-dimethoxyflavone (8.2 μg/ml, 5.0 μg/ml), 2′-benzyloxy-5,7,8-trimethoxyflavone (5.9 μg/ml, 11.0 μg/ml), 2′-hydroxy-5,7-dimethoxyflavone (8.3 μg/ml 4.9 μg/ml) 2′-hydroxy-5-methoxyflavone (4.2 μg/ml, 2.7 μg/ml), 2′-hydroxy-5,7,8-trimethoxyflavone (9.8 μg/ml, 6.2 μg/ml), 2′-benzyloxy-5-hydroxyflavone (5.2 μg/ml, 3.6 μg/ml), and 5,2′-dihydroxyflavone (5.1 μg/ml, 4.0 μg/ml). Presence of 5-methoxy group potentiated the cytotoxic activity, while the existence of 7-methoxy group decreased the activity. 5-Hydroxy or methoxy, activates 4-carbonyl group, while 7-methoxy group deactivates the carbonyl group. From these observation it was concluded that the activation of carbonyl group at C-4 of a flavone is important for the enhancement of the cytotoxic activity. The presence of both 5-hydroxy and 2′-benzyloxy- or 2′-hydroxy group enhanced the antitumor activity; 2′-benzyloxy-5-hydroxy-7-methoxyflavone (T/C=144%), 5,2′-dihydroxy-7-methoxyflavone (T/C=132%), and 5,2′-dihydroxy-6,7,8,6′ tetramethoxyflavone (T/C=172%). 2′-Hexanoylation of 5,2′-dihydroxy-flavones did not improve the antitumor activity; 2′-hexanoyloxy-5-hydroxy-7-methoxyflavone showed T/C=132%, about the same as that of 5,2′-dihydroxy-7-methoxyflavone (T/C=130%)     

Determining proxodolol in blood serum using reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method has been developed for the determination of proxodolol in the blood serum and tested in rabbits. The HPLC measurements were carried out using a Phenomenex (LUNA) C₁₈ (5 μm) column eluted with acetonitrile-1 mM sodium pentylsulfonate (25: 75, v/v) mixture adjusted at pH 3.0 with phosphoric acid; eluent flow rate, 1 ml/min. The UV detector was tuned to 272 nm. Sample preparation: to 0.5 ml of blood serum in a 10-ml glass centrifuge tube is added 100 μl of 2.5 N NaOH and 4 ml chloroform; the mixture is shaken for 2 min and centrifuged at 3000 rpm for 10 minutes. The chloroform phase is separated and evaporated to dryness in a flow of nitrogen. The residue is dissolved in 100 μl of 1-mM sodium pentylsulfonate (pH 3); a 50 μl aliquot is introduced into the HPLC column. The proposed method is simple, rapid, sensitive, and has a good linearity. The average recovery of proxodolol is 80%. The drug detection limit is 0.015 μg/0.5 ml. The method is linear in the interval from 0.02 to 1.0 μg/0.5 ml with a correlation coefficient of 0.9996. The coefficient of variation is below 2% within a day (n = 5). The method was used for determining the parameters of proxodolol pharmacokinetics upon peroral administration in a dose of 40 mg in rabbits. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 2, pp. 44–46, February, 2006.     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

Development and evaluation of a direct sandwich enzyme-linked immunosorbent assay for the quantification of guinea-pig immunoglobulin e

The purpose of this study was to develop and evaluate a direct sandwich enzyme-linked immunosorbent assay (ELISA) for the immunoglobulin E (IgE) in serum and plasma from guinea pig using mouse monoclonal antibodies specific for guinea-pig IgE. Mouse monoclonal antibodies were raised against purified IgE protein. The ELISA was performed using a combination of two anti-IgE monoclonal antibodies. One antibody was labeled with horseradish peroxidase (HRP), and the other was coated on polystyrene wells. Purified guinea-pig IgE was used as the standard material. The validity of the ELISA was confirmed by precision, dilution, recovery, and interference tests. The range of detection was 3.1-800 ng of IgE mass per mL of serum and plasma. The intra- and inter-assay coefficients of variation were 4.6% and 5.7%, respectively, or less. The recovery test showed variation only between 92.1% and 111.8%, and the anticoagulants showed noninterference with the IgE assay. The mean serum IgE mass concentration in OVA-sensitized guinea pigs was 29438 ng/mL, and it was 48.6 ng/mL in normal guinea pigs. The present ELISA is useful and practical for specific measurement of the guinea-pig IgE, and it is surmised that it would be suitable for use in allergological and pharmacological research.     

Antidiabetic and antidyslipidemic activities of Cuminum cyminum L. in validated animal models

The ethanolic extract of seeds of Cuminum cyminum (C.c) was found to improve glucose tolerance to the tune of around 18.3% (P < 0.01) in normal rats and shows around 17.7% (P < 0.01) and 17.1% fall on blood glucose levels at 0–300 and 0–1440 min, respectively, on streptozotocin-induced diabetic rats at an oral dose of 250 mg/Kg body weight. The extract has also been found to improve around 26.7% (P < 0.01) glucose intolerance on 14th day post treatment in high fructose fed streptozotocin-induced diabetic rats. The extract was also found to have antidyslipidemic activity as evident by 21.04% (P < 0.01) decline in serum triglycerides, 22.7% (P < 0.01) decline in total serum cholesterol, and 16.9% of decline in serum LDL-C, respectively, along with 12.2% (P < 0.05) increase in serum HDL-C on high fat diet fed male Syrian golden hamster. The extract was also found inhibitory to eye lens aldose reductase (EC 1.1.1.21) with IC₅₀ value of 7.07 μg/ml as compared to the standard AR inhibiting compound Quercetin which showed IC₅₀ 2.35 μg/ml. The extract was also found inhibitory to α-glucosidase with IC₅₀ value of 100 μg/ml as compared to known drug Acarbose which showed IC₅₀ of around 25 μg/ml.     

Mass spectrometry identifies multiple organophosphorylated sites on tubulin

Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 °C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.     

Lack of interaction between orlistat and oral contraceptives

Objectives: Orlistat, a potent and selective inhibitor of gastrointestinal lipases, is designed for the treatment of obesity. A double-blind, randomised, placebo-controlled, 2-way crossover study investigated the possible influence of orlistat on the ovulation-suppressing action of combination oral contraceptives (OC). Methods: After an 8-day run-in prior to the first of two consecutive menstrual cycles (Day 1 was the first day of menstruation), two groups of 10 healthy women, 20–27 years of age and on a stable regimen with OCs, received either 120 mg orlistat t.i.d. or placebo t.i.d. on Days 1–23 of the first cycle, and, separated by a placebo washout period on Days 24–28, the alternative treatment on Days 1–23 of the second cycle. In both cycles, serum luteinizing hormone (LH) was measured on Days 12–16 and progesterone on Days 12, 16, 19–23. Results: The geometric means of time-averaged concentrations (Days 12–16 for LH and Days 19–23 for progesterone) in the cycles with orlistat and placebo, respectively, and the one-sided 95% confidence region for the mean in the cycle with orlistat were 1.92, 2.03 and < 2.23 IU l⁻¹ for LH and 0.147, 0.145 and < 0.176 μg l⁻¹ for progesterone. The one-sided 95% confidence region for the ratio (orlistat/placebo) of geometric means was < 1.06 for LH and < 1.11 for progesterone. Conclusion: During normal ovulation the peak serum concentration of LH is above 30 IU l⁻¹ around Day 14 of the cycle, and that of progesterone exceeds 3 μg l⁻¹ around day 21. The 95% confidence regions for the means, as well as all individual concentrations, were below these limits. It was concluded that orlistat did not influence the ovulation suppressing action of oral contraceptives. Received: 18 April 1995/Accepted in received form: 6 November 1995     

Effects of lead on thyroid functions in lead-exposed workers

Lead exposure is a common public health problem. Exposure to the metal can cause hematological, gastrointestinal, rheumatological, endocrine, neurological and renal problems in humans. However, effects on the thyroid gland are controversial. We retrospectively investigated thyroid function parameters in 65 adult males who had been occupationally exposed to lead. We then compared the findings with those of 60 male patients who had no history of lead exposure or thyroid abnormalities, who served as the control group. The mean ages of the lead-exposed workers and the controls were 34.3 ± 7.9 and 32.9 ± 6.6 years respectively. Blood lead levels in the lead-exposed workers were significantly higher than in the control group. The lead-exposed workers were assigned to one of three groups according to their blood lead levels, as follows: 40–59 μg/dl, 60–79 μg/dl, or 80 μg/dl and above. Thyroid Stimulating Hormone (TSH) levels in the 80 μg/dl and above group were significantly higher than in either the 40–59 μg/dl group or the 60–79 μg/dl group. However, TSH levels in the 40–59 μg/dl group did not differ significantly from those in the 60–79 μg/dl group. These results suggest that high levels of lead in the blood may affect thyroid physiology. Clinicians should be aware of the potential hazardous effects of lead on the thyroid, especially in patients who have been occupationally exposed to lead.     

Biotin sulfone as a new tool for synthetic oligosaccharide immobilization: application to multiple analysis profiling and surface plasmonic analysis of anti-Candida albicans antibody reactivity against alpha and beta (1-->2) oligomannosides

As a part of our glycoantigen synthetic program for diagnosis and basic analysis of yeast-related pathogenic mechanisms, a library of 1-->2 oligomannosides suitable for immunoanalysis was prepared. The use of biotin sulfone, an oxidized form of biotin, offers a convenient solution for both oligosaccharide synthesis and immobilization on microspheres and surface plasmon resonance sensors. The application of this new strategy for the analysis of anti- Candida albicans antibody response through multiple-analyte profiling technology (Luminex) and with surface plasmonic analysis using biotin tagged synthetic oligosaccharides on avidin coated surfaces was validated using monoclonal antibodies.     

Patent Evaluation: Biotinylated nucleotide analogues as inhibitors of TNF hyperproduction

Novelty: Nucleotide analogues having a photoreactive group and a biotinyl group which covalently attach to nucleotide dependent protein binding sites are provided. Protein structurefunction studies are significantly facilitated by receptor binding site directed labelling. The invention has particular application to the characterisation of the active sites of nucleotide dependent enzymes. The use of the method for detection and removal of undesired DNA or RNA from cells (e.g. AIDS viral RNA) is briefly described.

Biology: Analogue modified proteins are detected by avidin-linked peroxidase or alkaline phosphatase methods. The modified protein can be purified by strepavidin-linked or avidinlinked affinity chromatography. After elution of unmodified protein, modified proteins are released and collected from the avidin column by raising the pH to hydrolyze the ester linkage by which the biotin is bound to the ribose hydroxyl group.

Chemistry: The biotin radical is attached to the ribose moiety of the nucleotide through an ester linkage. Upon photo irradiation the biotinylated photoaffinity analogues covalently bond to the nucleotide binding site.     

Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures

The genotoxic potential of the carcinogenic mycotoxin ochratoxin A (OTA) has been investigated by means of an in vitro micronucleus assay, an endpoint for genotoxicity which has not been studied previously for OTA. OTA was found to induce dose-dependently micronuclei (MN) in cytokinesis-blocked binucleated ovine seminal vesicle (OSV) cell cultures, which had been treated with the mycotoxin (12–30 μM) for 6 h in medium containing 10% fetal calf serum. For comparison, OSV cells were treated with colcemid (0.02–0.06 μg/ml), or 4-nitroquinoline N-oxide (NQO; 0.5 μM), a typical aneugen and clastogen, respectively. All test compounds increased the frequency of MN in OSV cells, the highest level being induced by 30 μM OTA. When MN were characterized by indirect immunofluorescence microscopy using anti-kinetochore (CREST) antibodies, the majority of MN in colcemid-treated cells was CREST-reactive (>70% kinetochore positive); as expected, this fraction was <10% for the NQO-treatment group. In cells treated with OTA the fraction of kinetochore positive MN was similar (33–40%) to that observed in solvent controls (38%). These data indicate that OTA induces MN apparently by a mixed, although predominantly clastogenic mode of action. OSV cells lack monooxygenase activity but express high prostaglandin H synthase (PGHS) activity. When cells were treated with OTA in the presence of indomethacin (10 and 50 μM), a well known inhibitor of PGHS, the frequency of MN induced by OTA was not decreased, but rather increased. This indicates that metabolic activation of OTA by PGHS seems not to be required for genotoxicity. The increased MN induction in OSV cell cultures is most likely due to competition of indomethacin with OTA for binding to serum proteins thus raising the fraction of free mycotoxin. Received: 27 August 1996 / Accepted: 28 November 1996     

Potentiality of an organometallic labelled streptavidin—biotin system in metalloimmunoassay

Biotin labelled with a cymantrene moiety (cyclopentadienyl manganese tricarbonyl complex) is described for the first time. Because this metallo-biotin retains full recognition for the specific glycoprotein avidin (or streptavidin), the labelled streptavidin—biotin system is proposed for use in a solid-phase competition-type metalloimmunoassay in which bovine serum albumin (BSA) is used as a model of applications. Atomic absorption spectrometry is utilized for the detection of the cymantrene-labelled biotin.     

Synthesis of bifunctional saxitoxin analogues by biotinylation

Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety.