Hydrogen-bonding propensities of sphingomyelin in solution and in a bilayer assembly: a molecular dynamics study

Sphingomyelin is enriched within lipid microdomains of the cell membrane termed lipid rafts. These microdomains play a part in regulating a variety of cellular events. Computer simulations of the hydrogen-bonding properties of sphingolipids, believed to be central to the organization of these domains, can delineate the possible molecular interactions that underlie this lipid structure. We have therefore used molecular dynamics simulations to unravel the hydrogen-bonding behavior of palmitoylsphingomyelin (PSM). A series of eight simulations of 3 ns each of a single PSM molecule in water showed that the sphingosine OH and NH groups can form hydrogen bonds with the phosphate oxygens of their own polar head, in agreement with NMR data. Simulations of PSM in a bilayer assembly were carried out for 8 ns with three different force field parameterizations. The major physico-chemical parameters of the simulated bilayer agree with those established experimentally. The sphingosine OH group was mainly involved in intramolecular hydrogen bonds, in contrast to the almost exclusive intermolecular hydrogen bonds formed by the amide NH moiety. During the bilayer simulations the intermolecular hydrogen bonds among lipids formed a dynamic network characterized by the presence of hydrogen-bonded lipid clusters of up to nine PSM molecules.     

Keratinophilic fungi isolated from the air at Pavia

The presence of keratinophilic fungi was revealed by sampling the air of Pavia (Italy) from March 1981 to February 1982. The species isolated were: Chrysosporium indicum, Geomyces pannorum var.pannorum, Microsporum gypseum, Myceliophtora vellerea and Trichophyton terrestre. Several filamentous fungi tolerating high levels of cycloheximide were also recovered.     

Complexes of 2-acetyl-gamma-butyrolactone and 2-furancarbaldehyde thiosemicarbazones: antibacterial and antifungal activity

Cobalt, nickel, copper and zinc coordination compounds of two thiosemicarbazones with general composition ML₂ (L: monodeprotonated ligand corresponding to 2-acetyl-γ-butyrolactone thiosemicarbazone, HL¹, and 2-furancarbaldehyde thiosemicarbazone, HL²) and also complexes with general composition MCl₂(HL²) were synthesized (except [NiCl₂(HL²)] and [Co(L²)₂]). The interaction of CuCl₂ with HL² gave [CuCl(HL²)], a copper(I) complex. The ligands and metal complexes were characterized by IR, ¹H and ¹³C NMR spectroscopy, and magnetic susceptibility measurements. The crystal structure of [Ni(L²)₂] · 2dmso was determined and a trans-square planar coordination of the two κ²-N,S chelate rings forming polymeric strips through H-bonds with dmso was observed. Actually, in all the reported complexes both ligands behaved as κ²-N,S chelates, except in the case of [Co(L¹)₂] in which HL¹ is tridentate κ³-N,S,O. The antimicrobial properties of all compounds were studied using a wide spectrum of bacterial and fungal strains. The copper complexes of HL² were the most active against all strains, including dermatophytes and phytopathogenic fungi. Most of the studied compounds, especially [Cu(L¹)₂], presented good activity against Haemophilus influenzae, a very harmful bacterium to humans.     

Complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin. A study of their antimicrobial activity

Cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin (HL(1) and HL(2), respectively) were synthesized and characterized, being the crystal structures of HL(1), HL(2) and [Ni(L(1))(2)].2CHCl(3) elucidated by X-ray diffraction techniques. The in vitro antimicrobial activity of all these compounds was tested against several bacteria and fungi. HL(1)and its complexes exhibited a strong inhibition of the growth of Haemophilus influenzae (MIC 0.15-1.50microg/mL) and good antibacterial properties towards Bacillus subtilis (MIC 3-25microg/mL). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound inhibiting the growth of each strain. The antibacterial effectiveness was confirmed against a number of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Yeasts and moulds showed a low susceptibility, except the dermatophyte mould Epidermophyton floccosum that is inhibited at concentrations ranging from 6 to 50microg/mL. In general, the antimicrobial activity of the thiophene derivatives was greater than that of the isonicotinic analogues.     

Design, synthesis, and biological evaluation of new (2E,6E)-10-(dimethylamino)-3,7-dimethyl-2,6-decadien-1-ol ethers as inhibitors of human and Trypanosoma cruzi oxidosqualene cyclase

New dimethylamino truncated squalene ether derivatives containing a different aromatic moiety (phenyl, naphthyl, and biphenyl) or a simple alkyl (n-hexylic) group were synthesized as inhibitors of the oxidosqualene cyclase (OSC) and of the sterol biosynthetic pathway. The activity against human OSC was compared with the activity against the OSCs of pathogenic organisms such as Pneumocystis carinii and Trypanosoma cruzi. The phenyl derivative was the most potent inhibitor of T. cruzi OSC.     

D-Asp: a new player in reproductive endocrinology of the amphibian Rana esculenta

We investigated the involvement of D-Aspartic acid (D-Asp) on ovarian and testicular morphology of the green frog, Rana esculenta, and its effect on the testosterone production. The study has been performed throughout the reproductive cycle. In both ovary and testis a substantial amount of D-Asp is endogenously present and its concentration varies as function of reproduction. In the frog, D-Asp content is differently correlated with gonadal and plasmatic levels of testosterone, depending on the sex. In fact, the amount of the D-Asp is inversely linked with that of the testosterone in the ovary, while this correlation directly matched in the testis. In vivo short-term experiments, consisting of a single intra-peritoneal injection of D-Asp (2.0 μmol/g body weight), demonstrated that the enantiomer is significantly accumulated by both the ovary and testis, reaching after 3 h the highest uptake and thereafter decreasing to baseline values within 24 h. Furthermore, D-Asp influences the synthesis and/or the release of testosterone, causing a decrease of its level in the female, and an increase in the male, respectively. In vivo long-term experiments, D-Asp, chronically administered to the frogs of both sexes, enhances the maturation of both gonads, determining in the oocytes an higher accumulation of carbohydrate yolk plates in the ooplasm, and stimulating the spermatogenesis in the testis. Taken altogether, our results show that D-Asp operates differently in female and male frog gonads, indicating that it has different targets in the reproductive machinery depending on the sex.     

The role of CXCR2 activity in the contact hypersensitivity response in mice

The recruitment of polymorphonuclear neutrophil leukocytes (PMN) into a challenge site, and their subsequent activation, are thought to play a role in the elicitation of the contact hypersensitivity (CHS) response. The present study investigated the role played by CXCR2 activity in tissue PMN infiltration and subsequent triggering of CHS. Our results show that the cutaneous infiltration by PMN, induced by hapten challenge was dramatically inhibited in sensitized, CXCR2-deficient (CXCR2(-/-)) mice. Inhibition of PMN recruitment into the hapten-challenged ears of CXCR2(-/-) mice was associated with a consistent reduction of the CHS response (ear swelling) in CXCR2(-/-) mice as compared with that observed in neutropenic, wild-type (CXCR2(+/+)) mice. Prevention of skin PMN infiltration and the ear swelling response by the absence of functional CXCR2 was observed regardless of the hapten used. These data clearly suggest that CXCR2 activity plays an essential role in mediating cutaneous recruitment and activation of PMN, and thus indirectly regulates recruitment of hapten-primed T cells into challenge sites, with the subsequent elicitation of the CHS response. The role played by CXCR2 activity in the CHS response provides the rationale for testing CXCR2 inhibitors as a new therapeutic approach to skin diseases.     

Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.     

Interactions of the C2 domain of human factor V with a model membrane

Activated coagulation Factor V is an important cofactor of the coagulation cascade that catalyzes the formation of the prothrombinase complex on the surface of membranes rich in phosphatidyl-L-serine (PS). Here we report molecular dynamics simulations of the two crystallographic structures (the open and closed conformations) of domain C2 of coagulation Factor V (FaVC2). The calculations were performed in water (1.5 ns for each conformation) and in the presence of a neutral phospholipid bilayer model (POPE; 10 ns for each conformation) in order to describe the dynamics of the free (plasma circulating) and membrane bound forms of FaVC2. Water simulations confirmed the hypothesis that the plasma circulating form is in the closed conformation. In contrast, the membrane simulations showed that both conformations are energetically compatible with membrane binding. We have investigated the mechanism, the dynamics, and the energetics of the binding process. Our data are consistent with published estimates of the immersion depth of the aromatic residues (W26 and W27), and with mutagenesis studies involving specific residues located on the spikes at the bottom of the FaVC2 structure. Electrostatic interactions between the phospholipid head groups and hydrophilic residues at the bottom of the structure play a key role in the binding process by creating a large number of hydrogen bonds that anchor the protein to the membrane. The simulations identified a stable phospholipid binding pocket reminiscent of a previously suggested PS interaction site. Our structural data could contribute to the design of potential inhibitors able to disrupt membrane association.