Optically detected magnetic resonance studies of porcine pancreatic phospholipase A2 binding to a negatively charged substrate analogue

The direct binding of porcine pancreatic phospholipase A2 and its zymogen to 1,2-bis(heptanylcarbamoyl)-rac-glycerol 3-sulfate was studied by optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. The zero-field splittings of the single Trp3 residue undergo significant changes upon binding of phospholipase A2 to lipid. Shifts in zero-field splittings, characterized mainly by a reduction of the E parameter from 1.215 to 1.144 GHz, point to large changes in the Trp3 local environment which accompany the complexing of phospholipase A2 with lipid. This may be attributed to Stark effects caused by the binding of a charged group near Trp3 in the enzyme-lipid complex. The cofactor, Ca2+, which is strongly bound to the enzyme active site, has an influence on the bonding, as reflected by smaller zero-field splitting shifts. A relatively small change in the Trp environment was observed for the interaction of the zymogen with lipid.     

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.     

Superoxide dismutase amplifies dye photosensitized production of desferal free radical: an electron spin resonance study

Desferal free radical (DFFR) photogenerated from dye sensitization was studied by electron spin resonance. When irradiated at the visible maximum in the presence of O2, both rose bengal and riboflavin sensitized the oxidation of Desferal (DF) and generated the DFFR. The yield of DFFR was amplified by superoxide dismutase (SOD). The SOD enhancement was attributed to the inhibition of superoxide-induced DFFR destruction. Similar SOD enhancement was observed with dyes Rhodamine 123 and Gentian Violet. Our studies suggest that when Desferal is used as a chelating agent in the presence of SOD, systems involving O2- could face interference from DFFR even at concentrations as low as 10 microM DF. DFFR may interfere with the chain reaction of lipid peroxidation resulting in an apparent protective action which, in fact, has very little to do with chelating the catalytic iron.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

Respiratory load compensation in neuromuscular disorders

First-breath ventilatory responses to graded elastic (delta E) and resistive (delta R) loads from 10 people with spinal muscular atrophy (SMA), 15 people with Duchenne muscular dystrophy (DMD), and 80 able-bodied people were compared. The SMA and DMD groups produced equal tidal volume, respiratory frequency, inspiratory duration (TI), expiratory duration, mean inspiratory airflow, and duty cycle responses to both delta E and delta R. Thus SMA (primarily a motoneuron disorder) and DMD (primarily a muscle disorder) have the same net effect on loaded breathing responses. The SMA and DMD groups failed to duplicate the normal group's short expirations during delta E, long inspirations during delta R, and thus, extended duty cycles during both delta E and delta R. The deficit in load compensation therefore was due to impaired regulation of respiratory timing (reflecting neural mechanisms) but not airflow defense (reflecting mechanical and neural mechanisms). One-fifth of the normal but none of the SMA or DMD subjects actively generated an "optimal" TI response (defined theoretically as TI greater than 160% control during large delta R and TI less than 75% control during large delta E). This lack of optimal responses, which is the same abnormality exhibited by quadriplegic people, suggests that SMA and DMD also impair human ability to discriminate between large delta R and delta E. These findings support the hypothesis that neuromuscular disorders can lead to disturbances in respiratory perception.     

Reduced bovine pancreatic trypsin inhibitor has a compact structure

The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.