Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly ¹³C/¹⁵N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive ¹³C/¹⁵N-labeled amino acids. The most cost-effective production of ¹³C/¹⁵N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% ¹³C-glycerol and 0.5% ¹⁵N-ammonium sulfate, supplemented with only 0.025% of ¹³C/¹⁵N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.     

Nutritional Status in the Experiment with 105-Day Isolation as the First Phase of the Mars-500 Project

In a 105-day experiment simulating crew life in a interplanetary spaceship, shifts in the nutritional status were assessed in six volunteersthat differed in the body weight index, basal metabolic rate, attitude to the proposed diet, physical exercise, and workload. The results of the investigation showed that hard physical work under the conditions of the experiment led to the formation of the nutritional status against the background of more intensive basal metabolism, elevated metabolism of carbohydrates and lipids, and their increased mobilization from fat depot. Food ration, though it was sufficient to sustain health and fairly high calorie, did not fully meet individual taste preferences of some crewmembers and energy needs for physical activities. Under these conditions, heavy workloads required mobilization of lipids from fat depot and reducedthe hepatic detoxification and metabolic capacities. Self-limitation of eating protein-rich desserts led to a relative deficiency of protein intake. These changes in the diet were the reason why four out of six test subjects reduced their basal metabolism and lost body mass. The recovery of metabolism and slowdown of the body weight loss were achieved under these conditions by supplementing meals with digestible proteincontaining products.     

Competitive binding of various beta-lactam compounds to penicillin-binding proteins of Escherichia coli

Competing interaction of two novel N-acyl derivatives of ampicillin i.e. N'-benzylchlorbenzimidazole (No. 48) and N-pyrazolytiazole (No. 72) derivatives and 14C-benzylpenicillin with penicillin-binding proteins (PBP) of E. coli was studied. It was shown that ampicillin and its derivative No. 48 markedly differed in their affinity to various PBPs. Derivative No. 72 did not prevent binding of the labeled benzylpenicillin to any PBP which corresponded to its low antimicrobial activity. Analogous experiments with new cephalosporin structures i.e. active and inactive N-acyl derivatives of cephalosporin showed that the active derivative No. 94 i.e. N-methyltiobenzimidazole derivative had the highest affinity to PBP-2 and PBP-5. The inactive derivative No. 68 i.e. N-chlorbenzimidazole derivative also had high affinity to PBP-1b, PBP-2 and PBP-3 essential for the cell. No activity of the latter compound against intact cells of E. coli was probably due to its low penetration through the outer membrane of the bacterial cell. Estimation of affinity of the beta-lactam structures to various PBPs not only provided data on the mechanism of their action but also made it possible to explain in some cases the peculiarities of their antimicrobial spectrum.     

Magnetic field excitation during electron beam injection from the Intercosmos-25 satellite (APEX)

Results of active experiments on electron beam injection from the Intercosmos-25 satellite into the ionospheric plasma are presented. A quasistatic magnetic field and the VLF-wave magnetic component are excited when an unmodulated electron beam with a current of I _be ?0.1 A and energy of ? _be =mv ²/2?10 keV is injected into the ambient plasma. The magnetic field excitation is attributed to the onset of plasma gradient instabilities.     

A critical evaluation of the conformational requirements of fusogenic peptides in membranes

It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an α-helical or a β-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF₃-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a β-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form β-structures due to sterical hindrance. If an α-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and – most importantly – (ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured α-helical peptide conformation or a β-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity. Dedicated to Prof. K. Arnold on the occasion of this 65th birthday.     

The dopamine system in rat brain areas in the early period following irradiation with superhigh doses]

In studying the main indices that characterize the neurochemical system of biosynthesis and degradation of a dopamine neuromediator, tyrosine hydroxylase-dopamine-monoamine oxidase, in different brain regions 5-6 min, 1 and 18 h after whole-body irradiation with high energy electrons (100 Gy) the authors have revealed a 25-40% inhibition of tyrosine hydroxylase and monoamine oxidase activity, and a 40% increase in the dopamine content of basal ganglia of the brain that control behavioural reactions of the organism. The neurochemical disturbances revealed are involved in the mechanisms of early transient incapacity after irradiation with superhigh doses.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR

The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the alpha-helix was determined by highly sensitive solid-state NMR measurements of (19)F dipolar couplings on CF(3)-labeled side chains, and supported by a nonperturbing (15)N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (>/=1:50 molar ratio) the helix axis changes its tilt angle from approximately 90 degrees to approximately 120 degrees , with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.     

Association of the ACE I/D gene polymorphism with DNA damage in hypertensive men

The aim of the study was to evaluate the association between the angiotensin-converting enzyme ACE I/D (rs 4340) polymorphism and DNA damage in patients with essential hypertension (EH). The I/D polymorphism of ACE was determined by polymerase chain reaction in 170 male hypertensive patients and 64 normotensive blood donors. We used flow cytometry to determine the levels of cell death, micronuclei and accumulation of peripheral blood leukocytes in G1/G0, S, G2/M phases of the cell cycle. Additionally, the whole blood samples were incubated in vitro at 4°C for 24 h to investigate the genotype effects on the susceptibility of cells to DNA damage. We found lower frequency of cells in DNA synthesis S phase and higher levels of micronuclei in the hypertensive compared to normotensive group (p < 0.05); increased formation of micronuclei was seen due to elevated micronuclei frequencies in patients with the ACE II genotype (p < 0.05), but not in ID or DD genotype carriers. Incubation of whole blood samples of normotensive individuals lead to the most active cell death (p < 0.05) and micronuclei formation (p > 0.05) in the II genotype carriers too. However, hypertensive patients displayed different cellular response to incubation-induced DNA damages in the ACE I/D genotype groups; after incubation, the frequencies of micronuclei were significantly higher in the DD genotype carriers (p < 0.05). To conclude, the study suggests that the ACE I/D polymorphism may contribute to mechanisms and intensity of DNA damages in hypertensive and normotensive individuals.