Solubilization of binding sites for IAPP and CGRP from rat lung

Functional binding sites for [¹²⁵I]IAPP and [¹²⁵I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (K_i = 22.9 nM) for [¹²⁵I]IAPP binding and rat CGRP (K_i = 0.904 nM) had a higher affinity for [¹²⁵I]CGRP binding over related peptides. [¹²⁵I]IAPP binding was unaffected by GTPγS, but [¹²⁵I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites.     

Binding of sex steroid binding protein to plasma membranes of human testis

The existence of a specific binding site for sex steroid binding protein (SBP or SHBG) was detected on plasma membranes prepared from the testis of a patient affected by a variant form of testicular feminization. A binding technique using [¹²⁵I]SBP as a tracer allowed us to identify a single set of binding sites, characterized by a K_d of 1.917 × 10⁻¹¹ M. The maximum number of binding sites was 5.2 fmol/mg membrane protein. Membranes were also prepared from a sample of genital skin from the same patient, but no binding for [¹²⁵I]SBP was detectable. The evidence of the SBP membrane receptor in the testis of a patient affected by Morris syndrome extends our knowledge about the tissue distribution of the SBP receptor and suggests the possible implication of SBP and its recognition system in a disorder related to peripheral androgen insensitivity.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Displacement of [H]GABA binding to bovine brain receptors by sulfur-containing analogues

The displacement of [³H]GABA binding to GABA receptors of bovine brain cortical membranes by some sulfur-containing compounds (homothiotaurine, thiotaurine and carboxymethylcysteamine) was investigated and their potency was compared to that of other known sulfur-containing analogues of GABA, such as homotaurine, homohypotaurine and taurine. Displacement studies showed homotaurine to be more effective as a GABA displacer than homohypotaurine and homothiotaurine (IC₅₀: 3.9 × 10⁻⁸, 6.7 × 10⁻⁷ and 6.8 × 10⁻⁷ M, respectively). Saturation experiments showed that the effect of taurine, homothiotaurine, homotaurine and homohypotaurine was due to a loss of high-affinity GABA sites (K_d = 10.7 nM). Homotaurine seems also to interact with low-affinity sites, decreasing the affinity constant, whereas the number of binding sites remains unchanged.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

New herbicide-binding site in the photosynthetic electron-transport chain: Competitive herbicide binding at the photosystem I phylloquinone-(vitamin K1)-binding site

The binding of herbicides to the phylloquinone-(primary electron acceptor A₁)-binding site in green plant photosystem (PS) I reaction centers is shown. Dissociation constants (K_d) of various herbicides to the phylloquinone-binding site were estimated by analyzing their competitive inhibition of the reconstitution of the phylloquinone analogue, menadione (vitamin K₃), to the phylloquinone-extracted spinach PS I particles. The phylloquinone-binding site was found to bind o-phenanthroline (K_d = 1.2 × 10⁻⁴ M), but only weak binding was observed with atrazine (K_d > 10⁻² M), although both are known to bind specifically to the quinone-(Q_B)-binding site in reaction centers of purple photosynthetic bacteria or PS II. The inhibitors of the cytochrome b/c₁(ƒ) complex, myxothiazol (K_d=9.5 × 10⁻⁶ M) or antimycin A (K_d = 2.8 × 10⁻⁶ M), also strongly bound to the phylloquinone site. This is the first report showing that the PS I reaction center complex also has a herbicide-binding site, although the site is probably not sensitive in vivo to these herbicides due to its higher affinity for phylloquinone than herbicides. The inhibitor specificity of the PS I phylloquinone site is different from that of the other quinone-functioning sites in the photosynthetic or respiratory electron-transfer chain, suggesting it to have a unique structure.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.     

Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (K_i < 1 × 10⁻⁹ M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (K_i = 6 × 10⁻⁸ M) > 5β-androstanedione > 5β-dihydrotestosterone (K_i = 3.5 × 10⁻⁷ M) > 5-androstane-3, 17β-diol (K_i = 5 × 10⁻⁶ M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10⁻⁴ M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.     

Receptor binding affinity and biological activity of C-terminal elongated forms of endothelin-1

Endothelin-1 (21 amino acids; ET-21) is considered to be derived from a precursor, proendothelin (38 amino acids; ET-38). In order to make the physiological significance of this conversion clear, we synthesized various C-terminal elongated derivatives of ET-21, such as ET-22, ET-23, ET-25, ET-31, ET-36 and ET-38 (each number implies the number of amino acid residues), and measured their receptor binding affinities and biological activities. When inhibition of [¹²⁵I]ET-21 binding to cultured rat smooth muscle cells (A10 cells) was measured, ET-21 inhibited with the highest affinity (IC₅₀ = 1.6 × 10⁻¹⁰ M) and the affinity of ET-38 was 30-fold less than that of ET-21. The binding affinities of the C-terminal elongated peptides were reduced with increasing number of amino acid residues, except for ET-22 whose affinity was lower than those of other peptides (IC₅₀ = 1.6 × 10⁻⁸ M). When contractions of rat aortic segments induced by these peptides were measured, ET-21 was the most potent (EC₅₀ = 2.8 × 10⁻¹⁰ M). All C-terminal elongated peptides, including ET-38, were more than 100-fold less active. It is noteworthy that ET-22 was the least potent peptide (EC₅₀ = 1.2 × 10⁻⁷ M). When bolus doses of C-terminal elongated peptides were administered to chemically denervated rats, the time-dependent change in blood pressure induced by each peptide was different from that induced by ET-21. Although ET-21 elicited a three phase depressor/pressor blood pressure response (an initial rapid hypotension, then a rapid transient hypertension followed by a slowly developing long-lasting hypertensive effect), the C-terminal elongated peptides, including ET-38, did not cause the initial transient hypotensive response. Very interestingly, the ability of the peptides to induce the rapid phase of hypertension in vivo does not seem to be correlated with the affinity of each peptide for the smooth muscle cell receptor, since the peptides with lower affinities for the smooth muscle receptor, such as ET-22, ET-23 and ET-25, showed more potent hypertensive effects. On the other hand, the slow and long-lasting hypertensive effect is likely to be related to the affinity of the compounds. The maximal hypertensive effects of cumulatively administered ET-21 derivatives were similar to those of ET-21. These results suggest that ET-21 is the most potent vasoconstrictor among the peptides and that the conversion from ET-38 to ET-21 may be important as an activation process.     

Ion-pair mechanism in square planar substitution. Reactivity of cationic platinum (II) complexes with negatively charged nucleophiles in solvents of high, medium and low polarity

The rates of displacement of dimethyl sulfoxide from the cation [Pt(phen) (CH₃) (Me₂SO)]⁺ by a series of uncharged and negatively charged nucleophiles have been measured in a methanol/water (19:1 vol./vol.) mixture. The starting complex and the reaction products were characterized either as solids or in solution by their IR and ¹H NMR spectra. The substitution reactions take place by way of a direct bimolecular attack of the ligand on the substrate. The sequence of reactivity observed is as expected on the basis of a nucleophilicity scale relevant for + 1 charged substrates ([Pt(en) (NH₃)Cl]⁺ used as standard). The difference of reactivity between the first (t-BuNH₂) and the last (SeCN⁻) members of the series spans five orders of magnitude. The value measured for the nucleophilic discrimination (1.55) is the highest found so far for cationic substrates. This is a result of the easy transfer of some of the electron density brought in by the incoming ligand into the ancillary ligands. When the reaction is carried out in a series of protic and dipolar aprotic solvents, using chloride ion as nucleophile, the rate of formation of [Pt (phen) (CH₃)Cl] is dominated by the extent of solvation of Cl⁻, as measured by its values of the Gibbs molar energy of transfer Δ_tG⁰. Conductivity measurements at 25°C in dichloromethane were fitted to the Fuoss equation and the values of the dissociation constants K_d for the ion pairs were calculated as follows: 2.27 × 10⁻⁵ M for Bu₄NCl, 2.75 × 10⁻⁵ M for Bu₄NSCN and 17.05 × 10⁻⁵ M for [Pt(phen) (CH₃) (Me₂SO)]PF₆. The pseudo-first-order rate constants k_obs for the reactions with Bu₄NCl, Bu₄NBr, Bu₄NSCN and Bu₄NI showed a curvilinear dependence on the concentration of the salt which levels off very soon (at concentrations higher than 0.005 M the kinetics are zero order in [Bu₄NX]). On addition of the inert electrolyte Bu₄NPF₆ the rates slow down and the kinetics follow the rate law k_obs = kK_ip[Bu₄NX]/[Bu₄NPF₆] + K_ip[Bu₄NX]). These findings fit well with a reaction scheme which involves a pre-equilibrium K_ip between ion pairs, followed by unimolecular substitution within the contact ion pair [Pt(phen) (CH₃) (Me₂SO)X]_ip. Values of the equilibrium constants K_ip for ion-pair exchange and of the internal substitution rates k were derived. The latter showed that the discrimination in reactivity between Cl⁻, Br⁻, SCN⁻ and I⁻ is greatly reduced with respect to aqueous solutions. The reason behind this may be desolvation of the ions coupled to the fact that a contact ion pair is already at a certain distance along the reaction coordinate in the direction of the transition state. Applications of the special salt effect and of ion pairing to synthesis are discussed.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca²⁺ and stimulated gastrin release in a dose-dependent manner over the range 10⁻⁵-10⁻³ M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-³H]scopolamine ([³h]nms) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (K_d = 39.5 pM, and B_max = 7.9 fmol/mg DNA). Carbachol competitively inhibited [³H]NMS binding. The potency of inhibition of [³H]NMS binding by subtype selective antagonists was hexahydrodifenidol> pirenzepine> AF-DX 116. These results suggest the M₃, muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.     

Dopamine receptors in human foetal brains: Characterization, regulation and ontogeny of [H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [³H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC₅₀ values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC₅₀ values of 2.5 and 10 μM, respectively suggesting a D₂ type receptor.

Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 10⁸ M⁻¹ min⁻¹ and 5.0 × 10⁻² min⁻¹, respectively. Equilibrium dissociation constant was 87 pM and the K_D obtained by saturation binding was 73 pM.

During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [³H]spiperone binding sites exhibited a heterogeneity with a high (K_D, 13–85 pM) and a low (K_D, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.

GTP decreased the agonist affinity as observed by dopamine competition of [³H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC₅₀ values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.     

Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities.

The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers.

With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03 ± 0.89 × 10⁻¹¹ mole/cm² of template from a 5.68 × 10⁻⁷ M myoglobin solution, compared to 2.58 ± 0.02 × 10⁻¹¹ mole/cm² for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 °C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal.

The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K_d) for the specific binding phase to be 3.4 × 10⁻⁷ M and the binding site capacity to be 7.24 × 10⁻¹¹ mole/cm². For the non-specific binding phase, K_d was found to be 1.355 × 10⁻⁵ M and the binding site capacity was determined as 9.62 × 10⁻¹⁰ mole/cm².

Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 × 10⁻⁷ M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.     

The short term effects of 5,7-dihydroxytryptamine on peripheral serotonin stores in Rhodnius prolixus and their long-term recovery

The serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) appears to affect invertebrate systems differently from vertebrate ones. The basis for toxicity in vertebrates appears to involve the intraneuronal actions of monoamine oxidase (MAO) upon the toxin. In insects, MAO is not present in appreciable amounts. In this study, we demonstrate that in vitro 5.7-DHT competitively inhibits the uptake of [³H]serotonin by serotonergic neurohaemal areas. The apparent K_M increases from 4.9 × 10⁻⁷ to 1.7 × 10⁻⁶ M. This neurotoxin also causes a significant release of previously accumulated [³H]serotonin in nominally Ca²⁺-free saline. While 5,7-DHT does not affect the uptake of [³H]tryptophan, it reduces the subsequent synthesis of [³H]serotonin. In vivo, the tissues appear to have recovered 2 weeks after toxin treatment, as determined by immunohistochemistry. At 24 h, 1 week and 2 weeks after injection, the tissues are able to take up and release [³H]serotonin normally. 1 and 2 weeks after injection, insects ingest a normal-sized blood meal, a behaviour acutely disrupted by 5,7-DHT treatment. The results of this and other invertebrate studies suggest that 5,7-DHT does not destroy serotonergic neurons, as it does in vertebrates. 5,7-DHT may be a more useful tool to study the functions of serotonin in invertebrates as one may transiently affect serotonin stores.     

Interaction of anthracycline antibiotics with biopolymers: 7. Equilibrium binding studies on the interaction of iremycin and DNA

We report spectrophotometric equilibrium studies of both the self-association of the new antibiotic iremycin and of its binding to calf thymus DNA in solution (ionic strength 0.2 M; pH 6.0). Iremycin forms dimers in this solution with a dimerization constant K₄=(1.19 ± 0.10) × 10³ M⁻¹. This equilibrium is taken into account in the evaluation of the interaction of iremycin with DNA. The binding behaviour can be completely described by a single binding mechanism of monomeric iremycin to DNA with allowance both for neighbour exclusion and for cooperativity of interaction. The three intrinsic binding parameters for the homogeneous model were determined simultaneously by a least squares fit of the original titration data: equilibrium constant of cooperative binding K = (2.72 ± 0.66) × 10⁵ M⁻¹ cooperativity parameter σ=0.38±3.27 ± 0.32. The binding parameters of iremycin and adriamycin and their microbial activities are compared.     

The presence of cortisol receptors in the human amnion

Cytosol extracts of human amnion tissue contained high affinity binding of cortisol (K_a = 2.48 ± 1.06 × 10⁹ M⁻¹; N = 30) and low capacity binding of cortisol (N_max = 279 ± 15.5 fmol mg⁻¹ protein). Kinetic studies of cortisol binding resulted in a similar value of K_a to that obtained by Scatchard analysis. Nuclear extracts of amnion tissue contained high affinity binding of cortisol (K_a = 5.8 ± 1.91 × 10⁷ M⁻¹) and low binding capacity (N_max = 91.4±21.4 fmol mg⁻¹ protein). K_a values were an order of magnitude higher in cytosol than in blood serum when amnion and blood were obtained from the same individuals. Differences in competitive ligand binding, especially dexamethasone, were observed between the amnion receptor and transcortin in serum. Gel permeation chromatography gave only one peak at 320 kDa for amnion receptor and only one peak at 48 kDa for transcortin from serum. When amnion tissue was incubated with or without cortisol, cytosol receptor activity was significantly lower in cortisol treated tissue than in control. The nuclear extracted receptor activity was significantly higher in cortisol treated tissue than control. The K_a values from cortisol treated tissue were significantly lower from control. Together the data support the presence of a specific cortisol receptor in the human amnion that is different from transcortin.