Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.     

Minimum inhibitory concentrations of herbal essential oils and monolaurin for gram-positive and gram-negative bacteria

New, safe antimicrobial agents are needed to prevent and overcome severe bacterial, viral, and fungal infections. Based on our previous experience and that of others, we postulated that herbal essential oils, such as those of origanum, and monolaurin offer such possibilities. We examined in vitro the cidal and/or static effects of oil of origanum, several other essential oils, and monolaurin on Staphylococcus aureus, Bacillus anthracis Sterne, Escherichia coli, Klebsiella pneumoniae, Helicobacter pylori, and Mycobacterium terrae. Origanum proved cidal to all tested organisms with the exception of B. anthracis Sterne in which it was static. Monolaurin was cidal to S. aureus and M. terrae but not to E. coli and K. pneumoniae. Unlike the other two gram-negative organisms, H. pylori were extremely sensitive to monolaurin. Similar to origanum, monolaurin was static to B. anthracis Sterne. Because of their longstanding safety record, origanum and/or monolaurin, alone or combined with antibiotics, might prove useful in the prevention and treatment of severe bacterial infections, especially those that are difficult to treat and/or are antibiotic resistant.     

Potentiating vanadium-evoked glucose metabolism by novel hydroxamate derivatives

L-glutamic acid () monohydroxamate (L-Glu()HXM) enhances the insulinomimetic activity of vanadium ions both in vitro and in vivo. Based on this ligand as a lead compound, and in order to delineate molecular features relevant to its anti-diabetic potential, 14 related derivatives, including short peptides, were synthesized by solution as well as by solid phase methodologies. In addition, hydroxamate derivatives of (+) pantothenic acid and D-biotin were prepared. The vanadium binding capacity of the hydroxamates synthesized was apparent, yet each had a different ligand-ions stoichiometry. The in vitro lipogenic potency of several compounds toward rat adipocytes was demonstrated. Thus, vanadium complexes of L-Gln()HXM, L-Glu()HXM-Gly, L-Aad()HXM, di-Glu-,-HXM and of (+) pantothenic acid hydroxamate exhibited 82, 79, 76, 39 and 39% of maximal insulin activity, respectively. L-Aad()HXM, L-Glu()HXM-Gly and (+) pantothenic acid hydroxamate – by themselves – were found to possess 24, 14 and 10% of maximal insulin activity, respectively. In vivo potency, however, of L-Gln()HXM vanadium complex in streptozocin-treated rat diabetic model was less apparent.     

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library

The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor alpha release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure--function analysis of lipoproteins in animal cells.     

Expression of a Plasma Membrane Proteolipid During Differentiation of Neuronal and Glial Cells in Primary Culture

Plasma membrane proteolipid protein (PM-PLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6, O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PM-PLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a seven-fold increase in PM-PLP synthesis with increases in biosynthetic increase in PM-PLP synthesis with increases in biosynthetic rate observed between days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM-PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 microM) was found to inhibit the appearance of radiolabeled PM-PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM-PLP to its target membrane. Colchicine (125 microM) also inhibited the appearance of newly synthesized PM-PLP in the plasma membrane by greater than 40%, suggesting that microtubules may also be required for PM-PLP transport to the plasma membrane.     

Differential regulation of sigma and PCP receptors after chronic administration of haloperidol and phencyclidine in mice

The existence of multiple receptor sites for the psychotomimetic agents phencyclidine (PCP) and some opiate-benzomorphans such as (+)N-allylnormetazocine ([+]SKF 10,047) in the mammalian central nervous system is well documented. These are: 1) sigma/PCP (sigma p) site, which binds both PCP and psychotomimetic opiates but not antipsychotics such as haloperidol, 2) PCP site, which selectively binds PCP analogs, and 3) sigma/haloperidol (sigma h) site, for which certain antipsychotics and (+)SKF 10,047, but not PCP analogs, display high affinity. In this study we examined the regulation of these receptor sites after chronic treatment of mice with either PCP or haloperidol. The following radiolabeled ligands were used to assess binding to the various receptor subtypes: [3H]-1-[1-[3-hydroxyphenyl)cyclohexyl]piperidine ([3H]PCP-3-OH; sigma p and PCP sites), [3H]thienyl-phencyclidine ([3H]TCP; PCP site), (+)-[3H]SKF 10,047 (sigma p and sigma h sites), and [3H]haloperidol (sigma h and D-2 dopamine receptors). Treatment of mice for 1, 7, 14, and 21 days with PCP (10 mg.kg-1.day-1) failed to induce variations in sigma p, sigma h, and PCP receptor binding. However, similar treatment with haloperidol (4 mg.kg-1.day-1) induced: 1) complete elimination of the binding to sigma h sites, 2) up-regulation of D-2 dopamine receptors, and 3) no change in sigma p and PCP receptor binding after 14 or 21 days of treatment. However, a single day of haloperidol treatment or in vitro incubation of mouse brain membranes with haloperidol failed to alter receptor binding. This study suggests that prolonged treatment of mice with haloperidol induces a loss in sigma h receptors that are presumably associated with certain psychotomimetic effects. This phenomenon is accompanied by an up-regulation of D-2 dopamine receptors.     

Metabolism of 15NH3 in Organotypic Cerebellar Explants and Cultured Astrocytes: Studies with Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry was used to study the metabolism of 15NH3 in organotypic cerebellar explants and cultured astrocyte monolayers. A steady-state level of 15NH3 was present by 1 min in both systems. Steady-state labeling in L-[amide-15N] glutamine, L-[15N]alanine, L-[15N]glutamate, and L-[15N]aspartate was attained by 1 min after 15NH3 addition in the organotypic cerebellar explants and by approximately 5 min in the cultured astrocytes. No measurable 15N labeling was noted in either glycine or serine in either system.     

Involvement of protein kinase C in the axonal growth-promoting effect on spinal cord neurons by target-derived astrocytes

Astroglial cells participate in a variety of developmental events during neuronal morphogenesis. We have shown that axonal, but not dendritic, outgrowth of spinal cord neurons can be promoted by a diffusible factor or factors secreted from target region-derived cerebellar astroglia in vitro in comparison with spinal astroglia. In the present study, we examined the involvement of protein kinase C (PKC) in the axon-promoting effect by astroglia. The inhibition of PKC by sphingosine or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) at high concentration greatly reduced the mean axonal length of spinal neurons cultured in medium conditioned by cerebellar astroglia (SCn-CBg), while activation of PKC by TPA at low concentration, or by retinoic acid, was not additive to the glial effect. The activation of PKC by TPA or retinoic acid promoted axon growth of spinal neurons cultured in medium conditioned by spinal astroglia (SCn-SCg), which otherwise would not be as supportive for axon growth as cerebellar astroglia. Western blotting and PKC activity assays showed that there was a trend for increased PKC activity and protein levels (in particular, PKCβ) in SCn-CBg cultures, which correlated with enhanced axon growth. Inhibition of PKC by sphingosine appeared to decrease protein levels, especially PKCβ, which correlated with suppressed axon outgrowth. In SCn-SCg cultures, phorbol ester activation of PKC increased both activity and protein levels of both PKCα and PKCβ. This activation correlated with stimulated axonal outgrowth. These results suggest that the glial signaling that regulates specific axonal outgrowth by target astroglia is mediated in part by the PKC second messenger system. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.