Competitive Displacement Assay Research Articles

Impairment of Methotrexate Transport Is Common in Osteosarcoma Tumor Samples

Osteosarcoma does not respond well to conventional dose methotrexate but does respond to high-dose methotrexate. Previous work has indicated that this resistance may be due to impaired transport of methotrexate across the cell membrane. In this study, the PT430 competitive displacement assay was adapted to evaluate methotrexate transport in 69 high-grade osteosarcoma tumor samples. All samples studied were shown to have relatively impaired methotrexate transport by PT430 assay. Ninety-nine percent of the samples had less than 20% PT430 displacement by methotrexate. Eighty-eight percent exhibited displacement by methotrexate at less than 50% of the displacement by trimetrexate. The high frequency of impaired transport suggests the presence of decreased functionality of the reduced folate carrier protein. The overwhelming presence of impaired transport may explain why methotrexate needs to be given in high doses to be effective in osteosarcoma therapy and suggests that reduced folate carrier-independent antifolates should be explored.

Structure-based investigation on the binding interaction of hydroxylated polybrominated diphenyl ethers with thyroxine transport proteins

Polybrominated diphenyl ethers (PBDEs) have been shown to alter thyroid hormone level in experimental animals. One of the possible mechanisms for hormone disruption is the competitive binding of hydroxylated PBDEs (OH-PBDEs) with hormone transport proteins. In this study, binding interaction of 14 diversely structured OH-PBDEs with two thyroxine transport proteins was investigated by fluorescence displacement assay, circular dichroism, and molecular docking. Binding affinity of the 14 OH-PBDEs with transthyretin (TTR) and thyroxine-binding globulin (TBG) was measured by competitive fluorescence displacement assay. The binding constant was found to fall in the range of 1.4 × 10 7 M −1 and 6.9 × 10 8 M −1 for TTR, and between 6.5 × 10 6 M −1 and 2.2 × 10 8 M −1 for TBG. Binding affinity increased significantly with bromination number from 1 to 4, whereas 5- and 6-brominated diphenyl ethers did not show any further increase. Protein secondary structural change of TTR and TBG upon binding with 5-OH-BDE-047 was investigated by circular dichroism. The spectral change displayed a pattern similar to the one with thyroxine, suggesting that the environmental chemical binds to the two proteins at the same sites as the hormone. In molecular docking analysis, a ligand-binding channel in TTR was revealed for OH-PBDEs binding, which appeared to be mostly hydrophobic inside but guarded by positively charged residue Lys15 at the entrance. Binding affinity of the 14 OH-PBDEs with TTR could be rationalized reasonably well by their pocket binding mode and hydrophobic characteristics. Based on the binding constant obtained in this work, possibility of in vitro competitive displacement of thyroid hormones from the transport proteins by OH-PBDEs was evaluated.

P63 Effect of pomegranate extract (PFE) in human radiodermatitis patients with squamous cell carcinoma (SCC) after radiotherapy

Methods Specific binding of LDE225 to human and mouse Smo protein was investigated in competitive displacement assays using a labeled small molecule Smo agonist (Hh-Ag 1.5). Inhibition of formation and induction of regression of basaloid tumors was determined in skin punch cultures derived from Ptch ± heterozygous knock-out mice. Inhibition of hair growth and Hh-target gene expression during early anagen phase was assessed after topical application of 0.3 or 1% solutions of LDE225 to depilated back skin of C57/BL6 mice twice daily for 7 days. Moreover, the penetration properties of LDE225 into and through human skin in vitro and pig skin in vitro and in vivo were tested.

Synthesis, cytotoxicity and DNA-binding of novel bisnaphthalimidopropyl derivatives in breast cancer MDA-MB-231 cells

New naphthalimidopropyl, bisphthalimidopropyl and bisnaphthalimidopropyl (BNIP) derivatives were synthesised and characterised. Their interactions with Calf Thymus DNA were studied by UV spectrophotometric analysis and a competitive Ethidium bromide displacement assay. Cytotoxicity was determined by MTT assay in a breast cell system (MDA-MB-231 and MCF-10A cells). All BNIPs exhibited strong DNA-binding properties and cytotoxic activity with IC 50 values in the range of 0.83–12.68 μM (24 and 48 h treatment). In addition, the uptake of BNIP derivatives within cancer cells was not via utilisation of the MGBG polyamine transporter. Put together the results confirm that the presence of the bisnaphthalimidopropyl and alkyl linker functionality are crucial for exerting DNA-binding and cytotoxic properties, hence demonstrating promise in their further development as potential anti cancer agents.

Ligand Binding Turns Moth Pheromone-binding Protein into a pH Sensor

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

Impairments in antifolate transport are common in retinoblastoma tumor samples

Many patients with retinoblastoma have a genetic predisposition to cancer and external beam radiation therapy and alkylating agent chemotherapy may increase their risk of secondary malignancy. Identification of effective chemotherapy agents for retinoblastoma that are not associated with an elevated risk of secondary malignancy would be beneficial. Twenty-six specimens of fresh retinoblastoma tumor cells were studied in vitro with a PT430 competitive displacement assay. Differential displacement of the PT430 by methotrexate and not trimetrexate was considered indicative of a defect in reduced folate carrier (RFC)-mediated transport. Elevations in the accumulation of PT430 were considered indicative of dihydrofolate reductase (DHFR) amplification. In 9 of the 26 (35%) samples, displacement by methotrexate was less than half the displacement by trimetrexate indicative of a defect in the RFC. In 5 of the 26 (19%) samples, trimetrexate did not displace the PT430. In 7 of 26 (27%) samples, the peak PT430 accumulation was suggestive of DHFR overexpression. Overall 9 of 26 (35%) samples had no evidence of a transport defect or DHFR overexpression and would be anticipated to be potentially sensitive to methotrexate. In 15 of the 26 (58%), no defects existed in trimetrexate displacement or DHFR overexpression and would be anticipated to be potentially sensitive to trimetrexate. These results would support consideration of a phase II study to determine the effectiveness of trimetrexate for recurrent intra-ocular retinoblastoma.

Staurosporine-based binding assay for testing the affinity of compounds to protein kinases

Staurosporine is a broad-spectrum inhibitor of both tyrosine and serine/threonine protein kinases. Excitation of staurosporine and its analogues at 296 nm results in major emission bands centered at 378 and 396 nm. The intensity of the emission bands is enhanced on binding to the adenosine triphosphate (ATP) site of many protein kinases. This property was used to develop a competitive displacement assay for evaluating the binding affinity of small molecules to protein kinases. The assay was validated in both cuvette and plate formats for several phosphorylated and non-phosphorylated protein kinases. The throughput of the assay is high enough to be used in drug discovery for screening as well as lead optimization.

Complementation Analysis Demonstrates That Insulin Cross-links Both α Subunits in a Truncated Insulin Receptor Dimer

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.

Modified-RNA Aptamer-Based Sensor for Competitive Impedimetric Assay of Neomycin B

The first label-free modified RNA-aptasensor for the detection of small molecules in biological samples is presented. A competitive displacement assay was applied to the detection of aminoglycoside neomycin B in whole milk using a fully 2‘-O-methylated RNA aptamer with faradaic impedance spectroscopic (FIS) detection. Neomycin B in solution displaces the aptamer from its complex with the SAM-immobilized neomycin B. The reusable aptasensor is capable of discriminating neomycin B from paromomycin, which differs from it in the substitution of a single amine group with a hydroxyl one. We show that the modified endonuclease-resistant RNA aptamer maintains the exquisite selectivity of the natural aptamer and allows the examination of biological samples of high protein content. This detection scheme can be readily adapted to the detection of other aminoglycosides and small molecules.

The L1Tc C-Terminal Domain from Trypanosoma cruzi Non-Long Terminal Repeat Retrotransposon Codes for a Protein That Bears Two C2H2 Zinc Finger Motifs and Is Endowed with Nucleic Acid Chaperone Activity

L1Tc, a non-long terminal repeat retrotransposon from Trypanosoma cruzi, is a 4.9-kb actively transcribed element which contains a single open reading frame coding for the machinery necessary for its autonomous retrotransposition. In this paper, we analyze the protein encoded by the L1Tc 3' region, termed C2-L1Tc, which contains two zinc finger motifs similar to those present in the TFIIIA transcription factor family. C2-L1Tc binds nucleic acids with different affinities, such that RNA > tRNA > single-stranded DNA > double-stranded DNA, without any evidence for sequence specificity. C2-L1Tc also exhibits nucleic acid chaperone activity on different DNA templates that may participate in the mechanism of retrotransposition of the element. C2-L1Tc promotes annealing of complementary oligonucleotides, prevents melting of perfect DNA duplexes, and facilitates the strand exchange between DNAs to form the most stable duplex DNA in competitive displacement assays. Mapping of regions of C2-L1Tc using specific peptides showed that nucleic acid chaperone activity required a short basic sequence accompanied by a zinc finger motif or by another basic region such as RRR. Thus, a short basic polypeptide containing the two C(2)H(2) motifs promotes formation of the most stable duplex DNA at a concentration only three times higher than that required for C2-L1Tc.

Design and synthesis of heterobimetallic donor–acceptor chemodosimetric ensembles for the detection of sulfhydryl-containing amino acids and peptides

A competitive indicator displacement assay has been successfully developed for the ratiometric determination of sulfhydryl-containing amino acids and peptides using heterobimetallic donor-acceptor complexes as chemodosimetric ensembles. Chromotropic cis-[ML2(CN)2](M = FeII, RuII, OsII; L = diimine) are used as signaling indicators and PtII(DMSO)Cl2 acceptor moiety is used as the receptor for the sulfhydryl-containing analytes. A series of three heterobimetallic donor-acceptor complexes: cis-FeII(bpy)2[CN-PtII(DMSO)Cl2]2 (1), cis-Ru(II)(bpy)2[CN-PtII(DMSO)Cl2]2 (2) and cis-Os(II)(bpy)2[CN-PtII(DMSO)Cl2]2 (3) are synthesized and characterized by X-ray crystallography. All the three ensembles are able to produce specific colorimetric/fluorimetric responses to sulfhydryl-containing amino acids (cysteine, homocysteine and methionine) as well as the sulfhydryl-containing small peptide glutathione. The mechanism of the competitive displacement assay is evaluated by examining the thermodynamics of formation of the donor-acceptor linkage and adducts between the acceptor metal and the sulfhydryl-containing analytes as well as by systematic variation of the donor and acceptor metals in the chemodosimetric ensembles.

Inhibition of Wild-Type and Mutant Human Immunodeficiency Virus Type 1 Proteases by GW0385 and Other Arylsulfonamides

The arylsulfonamide derivatives described herein were such potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K(i)) could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzyme concentration usable for the activity assay was much greater than the value of the dissociation constant). Consequently, two alternative methods were developed for estimation of K(i) values. The first method employed kinetic determinations of values for k(1) and k(-1), from which K(i) was determined (k(-1)/k(1)). The second method was a competitive displacement assay used to determine binding affinities of other inhibitors relative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [(3)H]GW0385 from E.[(3)H]GW0385. From the concentration of E.[(3)H]GW0385 at equilibrium, the concentrations of enzyme-bound and free inhibitors were calculated, and the ratio of the K(i) value of the unknown to that of GW0385 was determined (K(i,unknown)/K(i,GW0385)). The values of k(1) were calculated from data in which changes in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directly or indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complex formation were large enough to monitor directly. The value of k(1) for saquinavir was 62 +/- 2 microM(-1) s(-1). In the case of GW0385, the fluorescence changes associated with complex formation were too small to monitor directly. Consequently, the value of k(1) was estimated from a competition experiment in which the effect of GW0385 on the binding of E to saquinavir was determined. The value of k(1) for GW0385 was estimated from these experiments to be 137 +/- 4 microM(-1) s(-1). Because E.[(3)H]GW0385 was stable in the standard buffer at room temperature for greater than 33 days, the value of the first-order rate constant for dissociation of E.[(3)H]GW0385 (k(-1)) could be estimated from the time-course for exchange of E.[(3)H]GW0385 with excess unlabeled GW0385. The value of k(-1) calculated from these data was (2.1 +/- 0.1) x10(-6) s(-1) (t(1/2) = 91 h). The K(i) value of wild-type HIV-1 protease for GW0385, calculated from these values for k(1) and k(-1), was 15 +/- 1 fM. Three multidrug resistant enzymes had K(i) values for GW0385 that were less than 5 pM.

Identification and Characterization of the Hepatic Stellate Cell Transferrin Receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [(3)H]Thymidine incorporation, and real-time RT-PCR for procollagen alpha1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Influence of an asparagine to lysine mutation at amino acid 3516 of apolipoprotein B on low-density lipoprotein receptor binding

Background: Three mutations in the apolipoprotein B (apoB) gene have previously been established as important causes of impaired receptor binding of LDL and, hence, Familial Defective Apolipoprotein B 100 (FDB). Previously, undescribed mutations were sought. Methods: Using denaturing gradient gel electrophoresis for mutation detection, DNA from 1852 new patients was examined. Results: A previously undiscovered mutation was found in codon 3516, located between known FDB mutations at codons 3500 and 3531. The new mutation introduces a positively charged amino acid—lysine—while other FDB mutations remove a positively charged residue, arginine. The phenotype was intriguing, LDL derived from N3516K heterozygotes allowed only poor growth of an LDL cholesterol-dependent cell line. ApoB-100-specific antibody MB47 bound to LDL from N3516K heterozygotes with increased affinity indicating a probable conformational change caused by the substitution. In contrast to these results, a competitive displacement assay in fibroblasts showed normal (or better) binding affinity to LDL receptors and using dynamic laser scattering no preferential accumulation of 3516K LDL particles in plasma was found. Conclusion: Discovery of the mutation and characterisation of N3516K LDL reveals another naturally occurring apoB mutation that influences conformation of LDL apoB and its interaction with the LDL receptor.

Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon.

Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a "nucleic acid chaperone," promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription.

Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands

Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist in our diet or environment. This assay allows the binding of potential ligands to SHBG to be assessed under physiological conditions, and is sensitive to the effects of plasma constituents. Several classes of endocrine active compounds were tested, including hydroxy-polychlorinated biphenyls (HO-PCBs), phthalate esters, monoesters, chlorinated pesticides, as well as synthetic estrogens and phytoestrogens. The relative binding affinities (RBAs) of various compounds to SHBG were determined in competitive displacement assays, by comparison with 17β-estradiol (RBA=100). Synthetic estrogens bound SHBG with RBAs of 0.4 (ethinylestradiol)-0.2 (diethylstilbestrol), while some phytoestrogens bound with RBAs of 0.12 (coumestrol)-0.04 (naringenin). Many compounds did not bind to SHBG with sufficient affinity to allow RBA measurements, and these include: several phytoestrogens, such as genistein and kaempferol, polychlorinated biphenyls, phthalate esters and monoesters. Of nine HO-PCB congeners tested only 4-OH-2′, 3′, 4′, 5′-tetraCB and 4-OH-2, 2′, 3′, 4′, 5′-pentaCB bound SHBG in undiluted serum with RBAs of 0.05 and 0.11. Although all test compounds bound to SHBG with much lower affinity than endogenous sex steroids, these interactions may be physiologically relevant in situations where plasma SHBG levels are high and endogenous sex steroid levels are low, such as in pre-pubertal children and women taking oral contraceptives.

Mechanisms of methotrexate resistance in acute leukemia. Decreased transport and polyglutamylation.

Drug resistance limits the effectiveness of methotrexate (MTX) for the treatment of acute leukemia. An increased understanding of the pathways involved in folate metabolism has allowed investigations of the mechanisms of resistance observed in leukemic blasts obtained from patients. Acute lymphocytic leukemia (ALL) was studied for mechanisms of acquired MTX resistance. MTX transport in 27 patients with untreated ALL and 31 patients with relapsed ALL was measured using a previously described competitive displacement assay. Only 13% of the untreated patients were considered to have impaired MTX transport whereas over 70% of the relapsed patients had evidence of impaired MTX transport. Northern analyses and quantitative RT-PCR for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of 9 samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. Acute myelocytic leukemia (AML) was studied to determine the basis of the decreased MTX polyglutamylation. Enzyme kinetics of the enzyme folylpolyglutamate synthetase (FPGS) were studied, demonstrating FPGS in the myeloid cell lines and patient samples had a higher K(m) for MTX as a substrate than lymphoid cells. Measuring gamma-glutamyl hydrolase enzyme activity allowed a more accurate prediction of steady state levels of MTX polyglutamates. A knowledge of the mechanisms of MTX resistance that occur in leukemic blasts obtained from patients may allow the development of therapeutic strategies to circumvent resistance.

The goldfish vasoactive intestinal polypeptide receptor: functional studies and tissue distribution

Vasoactive intestinal polypeptide (VIP)1 is a neuropeptide that has numerous physiological actions and is widely distibuted in the body of most vertebrate species yet studied. A full-length VIP receptor cDNA was obtained by screening a goldfish brain and pituitary cDNA library. Functional expression of this receptor in mammalian COS-7 cells showed that it is coupled to cAMP production in a VIP (Atlantic cod, Gadus morhua) concentration-dependent manner; the EC50 of the VIP receptor in response to cod VIP stimulation was determined to be at 1.0 nM. At 100 nM of peptide, the relative potency of various peptides in stimulating cAMP in the transfected cells was cod VIP>human VIP>human PACAP-38>human PACAP-27>human secretin>dogfish glucagon>carp GHRH. The receptor was able to bind specifically to VIP and PACAP as indicated by competitive displacement assays. By reverse-transcriptase polymerase chain reaction, the transcripts of this receptor were detected in goldfish brain, pituitary gland, gall bladder, kidney, gill, small and large intestine, heart, muscle and spleen. The expression pattern of goldfish VIP receptor is similar to that of mammals and thus further evidence of the conservation of VIP function in vertebrates. Characterization of the VIP1 receptor from goldfish is the first step in understanding 2 of the most important neuropeptides, VIP and PACAP, and their interaction with multiple receptor system in lower vertebrates.

Cold acclimation increases fatty acid-binding protein concentration in aerobic muscle of striped bass,Morone saxatilis

Fatty acid-binding protein was isolated from aerobic skeletal muscle of the striped bass Morone saxatilis. FABP from striped bass (MS-FABP) has a molecular mass of 14,800 Da (as estimated by SDS-PAGE) and binds long-chain fatty acids with 1:1 molar stoichiometry and with micromolar affinity. Binding dissociation constants, as estimated by liposome assay, were 0.63 ± 0.44, 0.82 ± 0.12, and 1.60 ± 0.44 μM for palmitic (16:0), oleic (18:1), and palmitoleic (16:1) acids, respectively. No significant difference was detected in MS-FABP's binding affinity among these fatty acids or those estimated by competitive displacement assay (22:6, 20:5, and 18:0). Acclimation of striped bass from 25°C to 5°C caused an increase in FABP concentration in aerobic skeletal muscle (5°C = 1.44 ± 0.12 mg/g wet weight; warm = 1.04 ± 0.11 mg/g wet weight). The magnitude of this increase matches the predicted decrease in diffusion coefficient of FABP at 5°C. Previous studies, however, report that fatty acid oxidation is not only maintained but increased in cold-acclimated fish. We speculate that FABP may increase flux of fatty acids through β-oxidation (in cold-acclimated fish) by an increase in the percent of FABP molecules carrying fatty acid. © 1996 Wiley-Liss, Inc.

Positive cooperativity among insecticidal scorpion neurotoxins

The insecticidal activity of scorpion neurotoxic polypeptides increased 5–10-fold with no apparent increase in mammalian toxicity when a combination of two toxins was injected. Synergistic combinations could be predicted from binding studies and competitive displacement assays. Our results indicate that simultaneous expression in baculovirus or other transgenic organisms of the synergistic combinations of insecticidal toxins may result in more potent insect-selective biopesticides.