Study of conformation structure and molecular interactions of rosamycin by infrared spectrum analysis

IR spectra of rosamycin and its solutions in inert (CCl4 and C2Cl4), proton acceptor (tetrahydrofuran, hexametapol and diethylamine) and proton donor (CHCl3 and CH3OD) solvents were studied at various concentrations (0.1 to 0.001 mol/l) and temperatures (20 to 100 degrees C) in the region of the vC = O and vOH absorption bands (1600-1800 and 3200 3650 sm 1). It was found that the absorption bands at 3480 and 3560 sm-1 observed in the spectra of rosamycin diluted solutions in the inert solvents referred to variations of vOH...N of the aminosugar fragment and to vOH...O = C of the ester group of the macrocycle. Bands at 1697 and 1717 sm-1 referred to vC = O of the ketone and aldehyde carbonyl groups and band at 1728 sm-1 referred to vC = O of the ester group whose carbonyl was involved in the C = H...HO intramolecular hydrogen bond. Intensity of vC = O band (1745 sm-1) of the free ester group was nought. However, it increased with using the proton acceptor solvents. OH...N and OH...O = C intramolecular hydrogen bonds stabilized rosamycin molecule conformation. Mechanism of rosamycin interaction with the proton donor and acceptor molecules was elucidated. It was shown that tertiary nitrogen was the center of rosamycin molecule protonation.     

Investigation of the mechanisms for stark broadening and shift of the spectral lines in a nonideal plasma

Abstrac A method for calculating the Stark broadening and shift of the spectral lines was developed based on a combination of the ion plasma model and new theoretical and computational methods for taking the Stark effect into account. Theoretical results are compared with experimental data on the broadening and shift of the spectral lines in a strongly nonideal plasma. __________ Translated from Fizika Plazmy, Vol. 30, No. 10, 2004, pp. 944–947. Original Russian Text Copyright ? 2004 by Denisov, Orlov, Fortov, Kulish, Gryaznov, Mintsev.     

Hydration of denatured and molten globule proteins

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of alpha-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.     

Dispersal of the flea Ctenophyllus hirticrus and spreading of plague epizooties in Gorny Altai

Gradual dispersion of an abundant flea species Ctenophyllus hirticrus specific to the Pallas's pika (the main plague carrier), is revealed in the Gorno-Altai natural plague focus on the territory, occupied by two populations of this lagomorph. Spreading of Yersinia pestis in these areas took place a short time later the rise of this ectoparasite's abundance. It is supposed that the colonization of these areas by C. hirticrus was one of the factors determined epizooties spreading within the focus and formation of new sites of stable Y. pestis preservation.     

The diploid genome sequence of an individual human

Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.     

Effect of taurine on the microvessel exchange function and adrenergic response of veins and arteries in the cat skeletal muscle

In cats anesthetized with Uretan and perfused with a constant blood volume, Taurine induced responses of neither arterial nor venous vessels of the skeletal muscle but increased the capillary filtration coefficient without any significant change of the capillary pressure in the skeletal muscle's microvessels. Taurine also increased both the constrictor and the dilatory responses of the arterial and venous vessels. The mechanism of the Taurine effects upon the smooth muscle elements of arteries and veins as well as upon proper mechanisms of capillary pressure control and capillary filtration coefficient, seems to be calcium-dependent.     

A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.Proteomics experiments typically involve the analysis of peptide mixtures obtained by the enzymatic digestion of proteomes that can be as complex as complete cell lysates (1, 2). Dynamic range of peptide abundances and the sheer number of peptides encountered in these mixtures require extremely sensitive and fast peptide detection and fragmentation (3). Although a first comprehensively identified and quantified proteome has recently been reported (4), further gains in instrumental performance are clearly needed to reduce overall measurement time, improve sequence coverage of identified proteins, and for the in-depth analysis of mammalian proteomes.Among many different instrumental formats (5), the combination of a linear ion trap (6) with a Fourier transform (FT)1 mass spectrometer has rapidly become a popular technological platform in proteomics because it combines the sensitivity, speed, and robustness of ion traps with the high resolution capabilities of FT instruments. The first implementation of this principle used an ion cyclotron resonance instrument with a 7T magnet as the high resolution device (7). Later, the Orbitrap^TM analyzer developed by Makarov was coupled to the LTQ, combining the linear ion trap with a very small and powerful analyzer (8–11).Here we describe a next generation linear ion trap-Orbitrap instrument with significant improvements in ion source transmission and with a new ion trap configuration. We show that this instrument, termed the LTQ Orbitrap Velos, is capable of much higher scan speeds compared with the current LTQ Orbitrap. Furthermore, we implemented more efficient ion extraction for the higher-energy collisional dissociation (HCD) cell (12). Due to this improvement and the 10-fold higher transmission of ions from atmosphere, high resolution and high mass accuracy MS/MS can now routinely be obtained at very high sensitivity and at scan speeds of up to 5 Hz acquisition rates. A related instrument, the LTQ-Velos, which does not contain the Orbitrap analyzer for high resolution measurements, has been described very recently (13).     

Consensus generation and variant detection by Celera Assembler

Motivation: We present an algorithm to identify allelic variationgiven a Whole Genome Shotgun (WGS) assembly of haploid sequences,and to produce a set of haploid consensus sequences rather thana single consensus sequence. Existing WGS assemblers take acolumn-by-column approach to consensus generation, and producea single consensus sequence which can be inconsistent with theunderlying haploid alleles, and inconsistent with any of thealigned sequence reads. Our new algorithm uses a dynamic windowingapproach. It detects alleles by simultaneously processing theportions of aligned reads spanning a region of sequence variation,assigns reads to their respective alleles, phases adjacent variantalleles and generates a consensus sequence corresponding toeach confirmed allele. This algorithm was used to produce thefirst diploid genome sequence of an individual human. It canalso be applied to assemblies of multiple diploid individualsand hybrid assemblies of multiple haploid organisms. Results: Being applied to the individual human genome assembly,the new algorithm detects exactly two confirmed alleles andreports two consensus sequences in 98.98% of the total number2 033 311 detected regions of sequence variation. In 33 269out of 460 373 detected regions of size >1 bp, it fixes theconstructed errors of a mosaic haploid representation of a diploidlocus as produced by the original Celera Assembler consensusalgorithm. Using an optimized procedure calibrated against 1506 344 known SNPs, it detects 438 814 new heterozygous SNPswith false positive rate 12%. Availability: The open source code is available at: http://wgs-assembler.cvs.sourceforge.net/wgs-assembler/ Contact: gdenisov{at}jcvi.org Associate Editor: John Quackenbush     

Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation

Understanding the detailed metabolic mechanisms of membrane-associated cytochromes P450 is often hampered by heterogeneity, ill-defined oligomeric state of the enzyme, and variation in the stoichiometry of the functional P450.reductase complexes in various reconstituted systems. Here, we describe the detailed characterization of a functionally homogeneous 1:1 complex of cytochrome P450 3A4 (CYP3A4) and cytochrome P450 reductase solubilized via self-assembly in a nanoscale phospholipid bilayer. CYP3A4 in this complex showed a nearly complete conversion from the low- to high-spin state when saturated with testosterone (TS) and no noticeable modulation due to the presence of cytochrome P450 reductase. Global analysis of equilibrium substrate binding and steady-state NADPH consumption kinetics provided precise resolution of the fractional contributions to turnover of CYP3A4 intermediates with one, two, or three TS molecules bound. The first binding event accelerates NADPH consumption but does not result in significant product formation due to essentially complete uncoupling. Binding of the second substrate molecule is critically important for catalysis, as the product formation rate reaches a maximum value with two TS molecules bound, whereas the third binding event significantly improves the coupling efficiency of redox equivalent usage with no further increase in product formation rate. The resolution of the fractional contributions of binding intermediates of CYP3A4 into experimentally observed overall spin shift and the rates of steady-state NADPH oxidation and product formation provide new detailed insight into the mechanisms of cooperativity and allosteric regulation in this human cytochrome P450.