Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.     

A G beta protein in the Drosophila compound eye is different from that in the brain

A G protein beta subunit gene (Gbe) is expressed only in the eyes of adult D. melanogaster. This gene was identified by probing a Drosophila head cDNA expression library with monoclonal antibodies to a previously characterized Drosophila G protein beta subunit (Gbb). Immunoblot and Northern analyses demonstrate that Gbe protein and mRNA is not present in Drosophila mutants that lack eyes. Immunocytochemical and in situ hybridization analyses further demonstrate that Gbe is expressed in the eyes but not in the brain, whereas Gbb is abundantly expressed in the brain. The Gbe product is approximately 45% identical to previously identified G beta subunits and defines a new G beta class. Its localization suggests a possible role in phototransduction.     

Monoclonal antibodies to a nitroxide lipid hapten

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG₁ antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·10⁶ M^?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·10⁸ M^?1. This monoclonal IgG₁ mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG₁ is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

Halogen chemistry of the red alga Bonnemaisonia

Complete accounts of the natural products chemistry of Bonnemaisonia nootkana, B. asparagoides, B. hamifera and Trailliella intricata are described. In contrast to the chemistry of the closely related alga Asparagopsis, Bonnemaisonia spp. do not produce halomethanes, but instead an array of C₇-C₉ halogen-containing ketones, alcohols and carboxylic acids. Biomimetic syntheses of these compounds suggest they are precursors and products of in vivo Favorsky rearrangements.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Phase Equilibria in binary mixtures of dimyristoylphosphatidylcholine and cardiolipin

Paramagnetic resonance spectra of the spin-label 2,2,6,6-tetramethylpiperidinyl-l-oxy have been used to study phase separations in binary mixtures of dimyristoyl-phosphatidylcholine and cardiolipin. Two different samples of cardiolipin were used: (i) One sample contained calcium ions at a mole ratio of calcium:cardiolipin = 1:2; the experimental data support the view that cardiolipin is present in the bilayer membrane as calcium ion linked dimers, (CL)2 Ca2+. (ii) A calcium-free sodium cardiolipin sample yielded remarkable spin-label partition data that were quite different from those obtained in the presence of Ca2+. In both cases the spin-label data provide evidence for compound formation and for fluid-fluid immiscibility in the bilayer membrane.     

Application of pattern recognition and feature extraction techniques to volatile constituent metabolic profiles obtained by capillary gas chromatography

The applicability of threshold logic units, a form of nonparametric pattern recognition, to the processing of metabolic profile data obtained by high-efficiency glass capillary column gas chromatography has been investigated. The test data included profiles of the volatile constituents of urine from normal individuals and from individuals with diabetes mellitus. A feature extraction algorithm allowed for dimensionality reduction and indicated the constituents most important in the normal versus pathological distinction. With an optimum number of dimensions, a normal versus pathological prediction rate of 93.75% was achieved. Gas chromatography—mass spectrometry was utilized to identify important profile constituents.     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.