Jerome L. Gabriel

Molecular modeling of archaebacterial bipolar tetraether lipid membranes

Membranes composed of glycerol dialkylnonitol tetraether (GDNT) lipids from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied by molecular modeling. GDNT membranes containing eight cyclopentane rings in the molecule are packed much tighter than those without rings. When containing eight cyclopentane rings, the beta-D-galactosyl-D-glucose head-group of GDNT runs almost parallel to the membrane surface. However, when containing no rings, the head-group is oriented perpendicular to the membrane surface. Using molecular dynamics calculations, we have also conducted comparative studies of membrane packing between GDNT and various non-archaebacterial membranes. Compared to gel state dipalmitoylphosphatidylcholine (DPPC) and gel state distearoylphosphatidylcholine (DSPC) bilayers, the GDNT membrane with eight cyclopentane rings has a more negative interaction energy, thus a tighter membrane packing, while the GDNT without rings is less tightly packed than gel state DSPC. Based on the calculated interaction energies, the GDNT membranes (with and without rings) are much more tightly packed than DPhPC (an ester-linked diphytanyl PC) and DPhyPC (an ether-linked diphytanyl PC) bilayers. This suggests that the branched methyl group in the phytanyl chain is not the major contributor of the tight packing of GDNT membranes. The biological implication of this study is that the cyclopentane ring could increase GDNT membrane thermal stability. This explains why the number of cyclopentane rings in archaebacterial lipid increases with increasing growth temperature. Perhaps, through the ring-temperature compensation mechanism the plasma membrane of thermoacidophilic archaebacteria is able to maintain a tight and rigid structure, consequently, a constant proton gradient between the extracellular (pH 2.5) and intracellular compartment (pH 6.5), over a wide range of growth temperatures.

Design, synthesis, and biological evaluation of 1-(4-sulfamylphenyl)-3-trifluoromethyl-5-indolyl pyrazolines as cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) inhibitors

A series of 20 novel 1-(4-sulfamylphenyl)-3-trifluoromethyl-5-indolyl pyrazolines were designed, synthesized, and screened in vitro for anti-inflammatory activity. These compounds were designed for evaluation as dual inhibitors of cyclooxygenases (COX-1 and COX-2) and lipoxygenases (LOX-5, LOX-12, and LOX-15) that are responsible for inflammation and pain. All pyrazoline molecules prepared are optically active and compounds that are more potent in COX-2 inhibitory activity (5a and 5f) were resolved by chiral column and each enantiomer was tested for cyclooxygenase inhibitory activity. Molecular modeling and comparison of molecular models of 5a enantiomers with that of celecoxib model shows that 5a (enantiomer-1) and 5a (enantiomer-2) have more hydrogen bonding interactions in the catalytic domain of COX-2 enzyme than celecoxib. Compounds 5a, 5e, and 5f showed moderate to good LOX-5 and LOX-15 inhibitory activity and this is comparable to that of celecoxib and more potent than rofecoxib.

Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

The kinetic parameters of NAD-specific isocitrate dehydrogenase from bovine heart were examined at levels of substrates and effectors approximating the concentrations reported for isolated intact heart mitochondria in different respiratory states. The effect of changing ADP/ATP ratios (with total adenine nucleotides constant at 8 mmol/L) on enzyme activity was measured at constant concentrations of the substrates magnesium D-isocitrate (0.10 mmol/L) and NAD+ (3.0 mmol/L), the positive effector magnesium citrate (1.0 mmol/L) and the negative effector NADPH (1.5 mmol/L) at pH 7.4. Enzyme activity increased with increasing ADP/ATP ratios as a result of activation by rising ADP concentrations and not due to decreasing inhibition by falling levels of ATP. Increasing ADP decreased the inhibition by NADPH, and this effect was enhanced by magnesium citrate and by free Ca2+. In incubation media containing all of the above effectors, the S0.5 for enhancement of activity by free Ca2+ was 10 to 20 mumol/L at ratios of total ADP/total ATP between 2.0 and 0.1. This value is in the range of intramitochondrial concentrations of free Ca2+,1 but it is appreciably larger than S0.5 of Ca2+ (0.6 to 1 mumol/L) for the enhancement of ADP activation, which was determined in the absence of other effectors. When both the NAD+/NADH and the ADP/ATP ratios were decreased, a further decline in activity was found. The effect of the decreasing NAD+/NADH ratio was due to inhibition by NADH (apparent I0.5 = 0.23 +/- 0.03 mmol/L) since NAD+ was saturating over the range examined.

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.

Inactivation of a common epitope responsible for the induction of antibody-dependent enhancement of HIV.

Background: The primary antigenic domain responsible for complement-mediated antibody-dependent enhancement (C′-ADE) of HIV and simian immunodeficiency virus resides in the principal immunodominant sequence of the transmembrane protein. Objective: To identify whether there are amino-acid residues common to the epitopes of the known enhancing human monoclonal antibodies (MAb), and to provide a structural model for this functional region present on the HIV envelope. Since our model predicts that this region is involved in the association of gp120 with gp41, this association was monitored for each mutant. Design: The binding of enhancing human MAb to point and deletion mutations within the enhancing domain was analyzed by two methods. The first analyzed binding to mutants expressed in COS cells: the second quantified the binding of four enhancing human MAb to each mutant gp160 versus wild-type control by enzyme-linked immunosorbent assay (ELISA). Methods: Site-directed mutagenesis was used to produce specific deletions and point mutants, which were expressed in COS cells. Binding of MAb 50–69 and V3-loop MAb 5F7 were visualized in the wild-type and each of the mutant constructs by immunohistochemistry. Quantitative evaluation of enhancing human MAb binding to each mutant versus wild-type was performed by ELISA. A model for the enhancing domain and its relationship to gp120 association with gp41 was provided by molecular dynamics and ligand docking methods. Results: All available enhancing human MAb known to bind to the principal immunodominant region of gp41 were unable to bind to deletions involving the disulfide loop, which in our molecular model provided the primary association site between gp120 and gp41. Point mutations in the loop blocked this association, but had a quantitatively smaller effect on the binding of the enhancing human MAb. A conservative W596Y mutation completely blocked the binding of all human MAb, but had no effect on gp120–gp41 association. Conclusions: A variety of mutations within the primary C′-ADE domain inhibit binding of enhancing human MAb as well as blocking the association of gp120 and gp41. A conservative W596Y mutation blocks binding of all enhancing human MAb with retention of gp120–gp41 association. These data are important to the design of vaccines in which the primary enhancing epitope is disarmed to prevent the subsequent induction of an amnestic response that could lead to viral enhancement of infection. The retention of the gp120–gp41 association is postulated to yield an immunogen similar to natural infection for both subunit and genetic vaccines.

Identification of Sulfhydryl-modified Cysteine Residues in the Ligand Binding Pocket of Retinoic Acid Receptor β

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARβ mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARβ is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARβ. However, Cys228 lies closer to the opening of the RARβ ligand binding pocket whereas Cys267 lies more deeply buried.

The effects of calcium and lanthanide ions on the activity of bovine heart nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase

Abstract (i) The activity of purified NAD-specific isocitrate dehydrogenase from bovine heart was stimulated by free Ca 2+ in the presence of ADP and subsaturating levels of magnesium isocitrate, but not in absence of ADP. However, Ca 2+ was not absolutely required for ADP activation. This was particularly apparent when free Mg 2+ was kept low (0.0024–0.020 m m ) and the substrate magnesium dl -isocitrate ranged from 0.07–0.25 m m . When kinetic constants were determined at pH 7.4 under these conditions and in the absence of ethylene glycol bis(β-aminoethyl ether) N,N′ -tetraacetate, Ca 2+ had little or no effect on K m (app) for ADP; the stimulation of rate by Ca 2+ was mainly due to increased V (app). With subsaturating ADP, there was an interdependence in the interaction of the enzyme with substrate and Ca 2+ . Thus, with ADP constant (0.30 m m ) the values of K m (app) for magnesium dl -isocitrate declined from 0.35 m m at zero Ca 2+ to 0.19 m m with saturating Ca 2+ without affecting V ; K m (app) for free Ca 2+ declined with increasing magnesium isocitrate to a limiting K m of 0.3 μ m . (ii) Ethylene glycol bis(β-aminoethyl ether)- N,N′ -tetraacetate, frequently used as a calcium buffer, inhibited enzyme activity with and without ADP. (iii) The enzyme was not inhibited by the calmodulin inhibitors trifluoperazine and chlorpromazine. Inhibition by lanthanide ions of the isocitrate dehydrogenase was competitive with magnesium isocitrate and not with respect to Ca 2+ . The values of K is (1.8 to 3.1 μ m ) for La 3+ , Yb 3+ , Gd 3+ , Eu 3+ , Tb 3+ , and Er 3+ were about two orders of magnitude smaller than K m for magnesium dl -isocitrate.

Differential Role of Homologous Positively Charged Amino Acid Residues for Ligand Binding in Retinoic Acid Receptor α Compared with Retinoic Acid Receptor β

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARs) and retinoid X receptors. Although it has been suggested that the ligand binding domains (LBDs) of RARs share the same novel folding pattern, many RAR subtype-specific agonists and antagonists have been synthesized demonstrating that the LBD of each RAR subtype has unique features. We have examined the role of several positively charged amino acid residues located in the LBD of RARα in RA binding. These results are compared with previously published data for the homologous mutations in RARβ. Lys227 of RARα does not appear to be important for RA binding or RA-dependent transactivation, whereas the homologous residue in RARβ, Lys220, plays an important synergistic role with Arg269 in these two activities. In addition, Arg276 of RARα, like its homologous residue Arg269 of RARβ, was found to play an important role in the binding of RA most likely by interacting with the carboxylate group of RA. However, the orientation of and electronic environment associated with Arg276 in RARα appears to be different from that of Arg269 in RARβ, thus contributing to the uniqueness of the ligand binding pocket of each receptor.

Induction of mucosal anti-HIV antibodies by facilitated transfection of airway epithelium with lipospermine/DNA complexes

BACKGROUND Expression of microbial protein sequences in eukaryotic cells transfected by transcriptional/translational permissive cDNA constructs can induce systemic humoral and cellular responses in vivo. Two methods of in vivo transfection have been described to date. One method uses large quantities of naked DNA injected into skeletal muscle. The second method uses relatively small quantities of DNA complexed to gold particles for bollistic penetration of the plasma membrane of keratinocytes. The major disadvantage of the bolistic method is that instrumentation is required which is not generally available. OBJECTIVES The objectives of this study were to determine whether the use of DNA complexed with a cationic lipopolyamine could reduce the quantity of DNA required to induce systemic humoral responses following muscle transfection and whether similar DNA/lipopolyamine complexes could induce mucosal humoral responses following airway exposure. STUDY DESIGN Balb/c mice were exposed by nasal aerosol or intramuscular inoculation to a mammalian transcriptional/translational permissive DNA construct containing the entire sequence for the HIV-1 envelope polyprotein. Experimental animals were further segregated by the number of exposures at 3-week intervals and whether the DNA was complexed to dioctadecylamidoglycylspermine (DOGS) at a 5:1 molar charge ratio of DOGS/DNA. RESULTS DOGS facilitated in vivo transfection of mouse muscle reduced the quantity of DNA required for a systemic humoral response to surface expressed HIV-envelope proteins by one order of magnitude. Exposure of airway mucosa to both 10 micrograms and 1 microgram quantities of DNA complexed to DOGS produced systemic humoral responses to HIV-envelope as well as mucosal antibodies in pulmonary and colonic epithelia. Molecular modeling of DOGS/DNA complexes at the 5:1 charge ratio used in this study indicates that the DNA component is not exposed to aqueous solvents and may be relatively resistant to degradation under common biological environments. CONCLUSION Facilitated transfer of DNA by DOGS to transcriptional/translational competent cells offers several distinct advantages to the use of DNA alone. Since significantly smaller amounts of DNA are required, the potential for the induction of antibodies against DNA itself lessens the likelihood for the development of a lupus-like syndrome. More importantly, however, is the apparent ability to transfect mucosal cells which results in the development of specific mucosal immune responses. This procedure may allow the development of general methods for the induction of mucosal immunity at the level of entry for mucosal pathogens without the disadvantages inherent in live attenuated vectors.

Inhibition and activation of bovine heart NAD-specific isocitrate dehydrogenase by ATP.

In the absence of added calcium, inhibition of NAD-specific isocitrate dehydrogenase by ATP occurred without ADP (I0.5 = 1.8 mM) and with 0.2 mM ADP3- (I0.5 = 1.0 mM) at subsaturating substrate concentrations at pH 7.4. Inhibition by ATP was competitive with NAD+ in the presence and absence of ADP and was not reversed by magnesium citrate. No reversal of ATP inhibition by free Ca2+ was observed in the presence of ADP (0.2 mM). However, when ADP was absent, increasing Ca2+ first caused progressive reversal of ATP inhibition followed by activation by ATP. Without ADP, the S0.5 for calcium activation was 80-140 microM at ATP concentrations between 0.6 and 3.0 mM. The S0.5 for ATP activation, in the absence of ADP, was 1.1 and 2.1 microM when free Ca2+ was held constant at 0.1 and 1.0 mM, respectively. As in activation by ADP, ATP decreased the S0.5 for magnesium isocitrate without affecting V. However, in contrast to ADP, the activation by ATP occurred without lowering the Hill coefficient for the substrate. GDP activated the enzyme at relatively high concentrations of Ca2+ but not without added Ca2+.