Factors in the Testing and Application of Algicides

A review is presented of some of the factors affecting the laboratory testing and practical applications of chemicals toxic to algae. The basic factor demonstrated is that the amount of chemical required to inhibit the growth of algae is dependent on the amount of algae present and not on the volume of water in which the algae are dispersed. It is shown how a chemical can be tested for algistatic or algicidal properties, thus enabling one to decide how best to apply a particular chemical. The selectivity of chemicals and the development of resistance in algae towards certain chemicals is demonstrated. Also, it is shown how certain algae can appear to be resistant to chemical treatments because of their growth habit or their production of extracellular products which affect the toxicity of added chemicals. With a better understanding of how various factors can influence the effectiveness of toxic chemicals, it is hoped that the selection of a chemical and method of application to a particular problem will be more successful.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Modulation of gating of a metabolically regulated,ATP-dependent K+ channel by intracellular pH in B cells of the pancreatic islet

Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K⁺ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH _i . (1) The time course of K⁺(ATP) channel activity during exposure to and washout of NH₄Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH₄Cl entry and inhibition of the Na _o ⁺ H _i ⁺ exchanger, resembles the time course of pH _i measured in other cell types that have been similarly treated. (2) Increasing pH _o over the range 6.25–7.9 increases K⁺(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H⁺ by the application of the K⁺/H⁺ exchanger nigericin. (3) Increasing pH _i over a similar range produces similar effects on K⁺(ATP) channels in inside-out excised patches exposed to small concentrations of ATP _i . The physiological role of pH _i in the metabolic gating of this channel remains to be explored.     

Isolation of hydrophobic and hydrophilic variants of Candida albicans

We have previously demonstrated that most isolates of C. albicans are hydrophobic when grown at room temperature (RT, ca. 22-24 degrees C) and hydrophilic when grown at 37 degrees C. Variants of our standard strain LGH1095 were isolated that are hydrophobic at 37 degrees C and hydrophilic at RT. After repeated phase partitioning with cyclohexane-water cell populations that were 6-16% hydrophobic at RT and 66-80% hydrophobic at 37 degrees C were obtained. Subsequent limiting dilution experiments provided clones which were more hydrophobic at RT or hydrophilic at 37 degrees C. These were then recloned until the resultant populations were consistently under 5% cell surface hydrophobicity (CSH) at RT or over 95% at 37 degrees C. Treatment with several detergents as well as sugars did not decrease the CSH of these cells. Lipase and several proteases also had no effect. When treated with trypsin at a concentration twice that used to lower CSH of normal cells to less than 5%, the hydrophobic variant only decreased in CSH by 50%. Both variants were capable of germinating, although at different levels depending on prior growth temperature. Sensitivity to the germination inhibitor morphogenic autoregulatory substance (MARS) was similar to that of the parent strain.     

Crystallization of the aspartylprotease from the human immunodeficiency virus, HIV-1

The aspartylprotease of the human immunodeficiency virus HIV-1 (NY5) has been crystallized in a form suitable for x-ray diffraction analysis. The crystals are tetragonal bipyramids and produce an x-ray diffraction pattern that exhibits the symmetry associated with space group P4(1)2(1)2 (or its enantiomorph, P4(3)2(1)2). The unit cell parameters are a = b = 50.3 A, c = 106.8 A, alpha = beta = gamma = 90 degrees; measurable diffraction intensities are observed to a resolution of 2.5 A. Density measurements indicate one molecule of 9,400 daltons/asymmetric unit. The symmetry of this space group could accommodate the proposed active dimer species of the protease if the 2-fold axis were coincident with one of the crystallographic 2-fold axes.     

Aesthetic results following partial mastectomy and radiation therapy

This study was undertaken to determine the aesthetic changes inherent in partial mastectomy followed by radiation therapy in the treatment of stage I and stage II breast cancer. A retrospective analysis of breast cancer patients treated according to the National Surgical Adjuvant Breast Project Protocol B-06 was undertaken in 57 patients from 1984 to the present. The size of mastectomy varied between 2 x 1 cm and 15 x 8 cm. Objective aesthetic outcome, as determined by physical and photographic examination, was influenced primarily by surgical technique as opposed to the effects of radiation. These technical factors included orientation of resections, breast size relative to size of resection, location of tumor, and extent and orientation of axillary dissection. Regarding cosmesis, 80 percent of patients treated in this study judged their result to be excellent or good, in comparison to 50 percent excellent or good as judged by the plastic surgeon. Only 10 percent would consider mastectomy with reconstruction for contralateral disease. Asymmetry and contour abnormalities are far more common than noted in the radiation therapy literature. Patients satisfaction with lumpectomy and radiation, however, is very high. This satisfaction is not necessarily based on objective criteria defining aesthetic parameters, but is strongly influenced by retainment of the breast as an original body part.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).