Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors.

A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.

Affinity chromatographic purification of antibody subsets depends on the mode of cross-linking of antigen to the affinity matrix.

Affinity columns for the separation of rabbit antibodies (Abs) to the purified nicotinic acetylcholine receptor (AcChR) from Torpedo californica electroplax were constructed in two ways: (1) by direct cross-linking of purified AcChR to cyanogen bromide activated Sepharose 4B, and (2) by first cross-linking a purified curarimimetic neurotoxin to cyanogen bromide activated Sepharose 4B, subsequently saturating with AcChR, and finally cross-linking the toxin to noncovalently bound receptors with bisimidate cross-linkers such as dimethyl suberimidate (DMS). Ab(AcChR) from pooled rabbit antisera were chromatographed in near stoichiometric proportions to the AcChR bound to the column to allow equilibrium selection of antibodies directed against sites which were not sterically hindered. The concept that columns of the second type subfractionate Ab(AcChR) was tested by analyzing Ab(AcChR) from column fractions with an ELISA and radioimmunoassay (RIA) procedure. The ELISA was constructed to that the exposed face of the AcChR was the same as that expected for columns of the second type, i.e. for the internal or cytoplasmic face. Enhanced ratios of ELISA to RIA measures of Ab(AcChR) reflected substantial purification of cytoplasmic face antibodies in DMS-cross linked columns. Total Ab(AcChR) was measured by RIA. Both types of columns gave substantial purification of tightly bound antibodies, i.e. those which were eluted with 3M potassium thiocyanate. Thirty-fifty percent of the IgG eluted was found in these fractions, which contained 2-6% of the total eluted protein. Approximately half of the total IgG present in these fractions represented specific IgG against AcChR. Both types of columns could be reutilized giving similar results; however, their efficiency was diminished.

Bis-catechol-substituted redox-reactive analogues of hexamethonium and decamethonium: stimulated affinity-dependent reactivity through iron peroxide catalysis.

Symmetrically bis-catechol-substituted analogues (1 and 2, respectively) of hexamethonium and decamethonium were synthesized and investigated as redox-activated affinity reagents toward the neurotoxin-binding sites of the nicotinic acetylcholine receptor (nAcChR), purified from Torpedo californica electroplax. These reagents bound to nAcChR with Kd = 1.8 x 10(-8) and 2.3 x 10(-7) M for 1 and 2, respectively. In the presence of a metal, Fe(II)/Fe(III), and peroxide, both reagents produced a rapid and efficient half-of-sites inactivation of neurotoxin-binding sites in the nAcChR in a concentration-dependent manner, which paralleled the extent of receptor binding of the reagents. In the absence of Fe(II)/Fe(III) peroxide, redox-dependent inactivation occurred for both 1 and 2 more slowly and only at concentrations much higher (10(3)-10(4) times) than those necessary to produce significant binding to nAcChR. However, receptor inactivation in the absence of added metal peroxide was still more efficient for 1 and 2 than observed previously for [(trimethylammonio)methyl]catechol (3), the prototypic redox-dependent affinity reagent after which 1 and 2 were patterned. Thus, the new reagents reported are expected to provide more efficient and selective conditions for redox-dependent inactivation at nAcChR and other macromolecular sites to which such reagents may be directed.

Inhibition and inactivation of presynaptic cholinergic markers using redox-reactive choline analogs.

Inhibition and inactivation of two presynaptic cholinergic "markers", choline acetyltransferase and high affinity choline transporter, has been investigated using inhibitors designed with a redox-reactive catechol tethered to a quaternary ammonium group. Two quaternary ammonium alkyl-substituted catechols, 3[(trimethylammonio)methyl]catechol (TMC, 1) and N,N-dimethylepinephrine (catecholine, 2) were shown to bind weakly and noncompetitively to bovine choline acetyltransferase yet inactivated the enzyme in a time course consistent with the involvement of early intermediates in the spontaneous oxidation of these catechols. Both agents also inhibited high-affinity choline uptake. The time course of TMC and catecholine spontaneous oxidation-dependent inactivation of high affinity choline uptake sites was slower than, if it occurred at all, the spontaneous degradation of measurable choline transport in synaptosomes. When compared with inhibition of uptake of other neurotransmitters, it was shown that catecholine demonstrated more selectivity than TMC toward inhibition of choline transport. Km (microM) and Vmax (pmol/min per mg of protein) were measured for high affinity transport of choline, dopamine, and serotonin and were observed to be Km = 2.04 +/- 0.31, Vmax = 22 +/- 1; Km = 1.4, Vmax = 53; and Km = 0.15, Vmax = 23, respectively, in good agreement with published literature values. Ki's (mM) for catecholine and TMC, calculated from experimentally determined IC50's, were for catecholine 0.13 +/- 0.06, 0.53 +/- 0.09, and 0.39 +/- 0.10, and for TMC 0.06 +/- 0.03, 0.09 +/- 0.03, and 0.09 +/- 0.08, for choline, dopamine, and serotonin transport, respectively. In vivo studies using catecholine suggest that this compound impairs learning ability associated with long-term memory. Thus, catecholine represents a lead compound in a potential series of redox-reactive choline analogs, which may become useful irreversible antagonists of the critical cholinergic macromolecular targets underlying cholinergic hypofunction in disorders such as Alzheimer's disease.

Electronmicroscopic investigation of the effects of biocides on Pseudomonas aeruginosa PAO bacteriophage F116.

Electronmicroscopy was used to observe morphological changes of the Pseudomonas aeruginosa PA0 bacteriophage F116 when treated with various biocides commonly used as antibacterial and antifungal agents. Because of its large size (145 nm) and its organised structure (an isometric head and a tail), it was possible to classify structural damage into eight categories. The morphological changes induced depended on the type of biocide used and its concentration. Glutaraldehyde increased the number of phages with empty heads. Peracetic acid and phenol altered the appearance of the viral genome packaged inside the head, produced fractured heads, and damaged the tail. Peracetic acid also induced folding of the phage heads. The alcohols tested also altered the head structure. Cetylpyridinium chloride induced mainly fractured head damage. Chlorhexidine had little effect on the structure of F116.

An enzyme-linked immunosorbent assay for antibody against acetylcholine receptor.

An enzyme-linked immunosorbent assay is described for measurement of antibody against Torpedo acetylcholine receptor. As here developed, the assay is highly sensitive, reproducible, and requires small quantities of immunological reagents. Relative measurements of antibody concentration by this method are proportional to those determined by radioimmunoassay.

The efficacy of the disinfection of bronchoscopes contaminated in vitro with Mycobacterium tuberculosis and Mycobacterium avium-intracellulare in sputum: a comparison of Sactimed-I-Sinald and glutaraldehyde.

The efficacy of Sactimed-I-Sinald and glutaraldehyde was tested against clinical isolates of Mycobacterium tuberculosis (MTB) and Mycobacterium avium-intracellulare (MAI). An in-use method demonstrated that over 10 disinfection cycles, using an auto-disinfector and a contact time of 60 min, MTB was eradicated in 10 out of 10 cycles with Sactimed-I-Sinald and five out of 10 cycles for glutaraldehyde. For MAI, Sactimed-I-Sinald showed a 5 log reduction at a 60 min contact time, which was not seen with glutaraldehyde. Although further evaluation is necessary, Sactimed-I-Sinald appears a promising alternative to glutaraldehyde.

Clinical correlates of enzyme-immunoassay versus radioimmunoassay measurements of antibody against acetylcholine receptor in patients with myasthenia gravis.

Antibody against human nicotinic acetylcholine receptor [Ab(AcChR)] was measured in the sera obtained from 55 patients with myasthenia gravis (MG) using both radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). By at least one assay, 91% of the patients had elevated Ab(AcChR). We found no correlation between the amount of Ab(AcChR) measured by RIA and that measured by ELISA. Patient subpopulations defined by ELISA- or RIA-measured Ab(AcChR) were associated with different disease durations. All of those who had high Ab(AcChR) levels by both assays had experienced symptoms for less than 2 years. 87% of those with high Ab(AcChR) levels by ELISA had had MG for less than 4 years. Those patients with high Ab(AcChR) only by RIA had a mean disease duration of over 8 years. With regard to correlations of Ab(AcChR) with patient age and sex, females under 50 years of age had high levels of Ab(AcChR) by RIA, but had lower levels by ELISA, whereas men over 50 had high Ab(AcChR) levels by ELISA. Using either assay, no relationship was established between concentrations of Ab(AcChR) and the patient's functional status, previous thymectomy, or current therapy. In this study, 16% of the MG patients with elevated Ab(AcChR) would have been considered within the non-disease range of Ab(AcChR) had only the RIA been performed, thus recommending the routine use of both assays for diagnostic purposes.

Use of erythrocyte hemolysis kinetics in the purification of complex cardiotoxin mixtures.

A human erythrocyte hemolysis kinetic method provides a useful way to follow the purification of cobra venom cardiotoxins or other hemolytic factors. Initial rates of hemolysis, measured as hemoglobin released with time for the separated cardiotoxins from the venom of the Thailand cobra Naja naja siamensis, vary over a greater range than do other commonly used measures of their biological activity. Recovery of the hemolytic activity of the gross cardiotoxin fraction in the subsequently separated fractions has been demonstrated. The method employs submilligram quantities of mixed or purified cardiotoxins.

Effects of cardiotoxin D from Naja naja siamensis snake venom upon murine splenic lymphocytes.

Cardiotoxin D from Naja naja siamensis is cytotoxic to T-lymphocytes above 150 femtomoles/10(6) cells. Equivalent lysis of macrophages or B-lymphocytes requires at least 1000 times more toxin. Reduction and carboxamidomethylation of cardiotoxin D does not effect T cell lysis. At higher toxin concentrations, 50% T-cell lysis occurs within 10 min. Splenocytes cultured with mitogens are up to five times more susceptible to toxin than unstimulated cells. Cardiotoxin D may directly disrupt the plasma membrane, since lysis is unaltered at 4 degrees C.

Separation of the enzyme cofactor pyrroloquinoline quinone and three isomeric analogues by capillary electrophoresis with ion-pairing media.

The enzyme cofactor pyrroloquinoline quinone (PQQ) was successfully separated from three closely related isomeric analogues by capillary electrophoresis with ultraviolet detection. Rapid and efficient separation of all four negatively charged isomers with baseline resolution was achieved by the addition of low concentrations (1-5 mM) of short chain tetraalkylammonium (TAA) salts to the capillary buffer. The TAA cations act as ion-pairing agents and promote differential migration of the isomers with only a minimal reduction in the electroosmotic flow. The effects of the TAA salt concentration and the alkyl chain length were examined. Detection limits of PQQ and its isomers were in the range of 7-15 microM with mass detection limits of 98-210 fmol.

Chiral separation of highly negatively charged enantiomers by capillary electrophoresis.

The separation of two highly negatively charged enantiomeric organic disulfates containing two chiral centers was investigated by capillary electrophoresis using cyclodextrin based chiral selectors added to the run buffer. The optimum separation for the enantiomers was achieved in less than 3 min at 25 degrees C with a run buffer of 10 mM glycine pH 2.4 and 5 mM QA-beta-CD, which is a positively charged quaternary ammonium beta-cyclodextrin derivative. The method resulted in baseline resolution, excellent linearity, and highly reproducible migration times allowing facile evaluation of the enantiomeric purity of the individual isomers. Detection limits for the enantiomeric pair were determined to be 0.3 ng/microl (S/N = 3). The nature of the selector-enantiomer interaction and a quantitative measurement of the apparent stability constants that governed chiral discrimination of the enantiomers with QA-beta-CD were also investigated by UV-Vis spectroscopy and electrospray ionization mass spectrometry.

Isolation and partial purification of phthalate ester hydrolyzing enzyme(s) from the brine shrimp, Artemia.

Isolation and 30-fold purification of a di-n-butyl phthalate (DNBP) hydrolyzing enzyme from synchronously hatched larvae of the brine shrimp, Artemia, are reported. The activity of the enzyme increases during larval development simultaneously with the acute toxic action of DNBP on the larvae. The enzyme is not stimulated by 10 mM sodium cholate, is stable for days to weeks in solution at 4 degrees C, indefinitely stable at -70 degrees C and is distinct from the previously reported trypsin- and chymotrypsin-like proteases [1].

Psychoanalytic theory and affirmation of the gay lifestyle: are they necessarily antithetical?

This article explores a conflict between the American psychoanalytic community and the American gay community which has played itself out in this country's mental health system for most of this century. The roots of the conflict are traced back to the American conception of diagnosis, as well as a lack of adequate developmental studies focused on the nature of homosexuality. The current psychoanalytic stance is presented as inimical to the stance Freud took, and an exploration of ways to ameliorate the conflict between American psychoanalytic thought and affirmation of homosexuality as an alternative healthy lifestyle is undertaken.

Synthesis and characterization of a heterobifunctional mercurial cross-linking agent: incorporation into cobratoxin and interaction with the nicotinic acetylcholine receptor.

The heterobifunctional organomercurial reagents 3-(acetoxymercurio)- and 3-(chloromercurio)-5-nitrosalicylaldehyde were prepared, characterized in model studies, and used to probe the interaction between cobratoxin, purified from the venom of the Thailand cobra (Naja naja siamensis), and the affinity-purified nicotinic acetylcholine receptor (AcChR) from Torpedo california electroplax. These reagents may also be useful in introducing chemically well-defined heavy metal atoms into proteins containing no reactive thiols. Model reagent adducts were prepared in situ by reductive amination with N-butylamine and N alpha-acetyllysine-N-methylamide. The nitrophenolic pKaS of the amine adducts were similar to those of the aldehyde reagents through reduced by 1.3-1.5 units when compared with the hydroxylmethyl reduction product. Reaction of either mercuriosalicylaldehyde with cobratoxin led to a single major modification product incorporating 1 mol of the reagent into cobratoxin at Lys 23. The Lys 23 modified toxin had a reduced binding affinity for the AcChR over that of the native toxin (Kd 2.75 nM cf. 0.3 nM). Reduction of the purified AcChR with 1 mM dithiothreitol (DTT) followed by removal of excess thiol led to cross-linking reactions with the Lys 23 modified cobratoxin to both the alpha and beta subunits of the AcChR complex. Reaction of DTT-treated AcChR with N-ethylmaleimide (NEM) blocked cross-linking, while treatment of the initially cross-linked toxin-AcChR complex with mercaptoethanol leads to reversal of cross-linking. These observations strongly support cross-linking mediated by the formation of a mercury-sulfur bond and further lend support the identity of the respective interacting sites in AcChR and toxin.