The Role of Influenza A Virus Hemagglutinin Residues 226 and 228 in Receptor Specificity and Host Range Restriction

Influenza A viruses can be isolated from a variety of animals, but their range of hosts is restricted. For example, human influenza viruses do not replicate in duck intestine, the major replication site of avian viruses in ducks. Although amino acids at positions 226 and 228 of hemagglutinin (HA) of the H3 subtype are known to be important for this host range restriction, the contributions of specific amino acids at these positions to restriction were not known. Here, we address this issue by generating HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions. We also let ducks select replication-competent viruses from a replication-incompetent virus containing a human virus HA by inoculating animals with 10^10.5 50% egg infectious dose of the latter virus and identified a mutation in the HA. Our results showed that the Ser-to-Gly mutation at position 228, in addition to the Leu-to-Gln mutation at position 226 of the HA of the H3 subtype, is critical for human virus HA to support virus replication in duck intestine.     

Alternative origins of stroma in normal organs and disease

Stromal fibroblasts are a new prospective drug target. Mesenchymal stromal cells (MSCs) and monocyte-derived stromal cells, also known as fibrocytes, are distinct fibroblastic populations derived from separate lineages. Mesenchymal and myeloid fibroblast progenitors are multipotent, serve as progenitor cells in animal models, and are implicated in several diseases. In addition, epithelial-mesenchymal transition (EMT) has been established as a mechanism for generation of stromal cells. Organ sources, relative contributions, and functions of these populations in normal development and pathology are not well understood. Innovative approaches are needed to identify markers that can distinguish these stromal populations.     

The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria

The contractile tail of bacteriophage T4 is a molecular machine that facilitates very high viral infection efficiency. Its major component is a tail sheath, which contracts during infection to less than half of its initial length. The sheath consists of 138 copies of the tail sheath protein, gene product (gp) 18, which surrounds the central non‐contractile tail tube. The contraction of the sheath drives the tail tube through the outer membrane, creating a channel for the viral genome delivery. A crystal structure of about three quarters of gp18 has been determined and was fitted into cryo‐electron microscopy reconstructions of the tail sheath before and after contraction. It was shown that during contraction, gp18 subunits slide over each other with no apparent change in their structure.     

Glutathione peroxidase 1 (<Emphasis Type=Italic>GPX1</Emphasis>) single nucleotide polymorphism Pro198→Leu: Association with life span and coronary artery disease

In this study, we genotyped polymorphism in GPX1 Pro198→Leu (C→T) rs 1050450 in four groups, i.e., patients with coronary artery disease, people who lived a long time (over 90 years), people who died early (before 55 years) from cardiovascular disease, and the Russian population as a control group. We have found a significant higher T-allele frequency in men with coronary artery disease, i.e., 34.84% (χ² = 5.228, p = 0.022; OR =1.46), and in men who died early from cardiovascular diseases, 38.16% (χ² = 6.461, p = 0.011; OR = 1.69) compared to men in the control group, 26.8%. Moreover, a significantly higher genotype TT frequency has been shown in patients with coronary artery disease and myocardial infarction before age 50, which is 19.44% compared to the control group, which was 7.28% (χ² = 9.55, p = 0.002). The TT frequency in individuals who lived a long time (4.39%) was the lowest and differed significantly from the group with coronary artery disease, which was 12.79% (χ² = 8.07, p = 0.0045), and from the subgroup with coronary artery disease with myocardial infarction before age 50, which was 19.44% (χ² = 14.49, p = 0.0001). Thus, our results indicate that the TT allele (Leu) of GPX1 Pro198→Leu (C > T) polymorphism is unfavorable for successful aging; it leads to predisposition to coronary artery disease, early myocardial infarction (before age 50), and early death (before age 55).     

Bioremediation of oil polluted aquatic systems and soils with novel preparation `Rhoder'

This paper summarises the experience accumulated duringthe field application of biopreparation `Rhoder' (solely or in a combinationwith preliminary mechanical collection of free oil) for remediation of oil polluted aquatic systems and soils in the Moscow region and Western Siberia during 1994–1999.It was demonstrated that `Rhoder' had a very high efficiency (>99%) for bioremediation of the open aquatic surfaces (100 m² bay of the River Chernaya, two 5,000 m² lakes in Vyngayakha) at initial level of oil pollution of 0.4–19.1 g/l. During remediation of the wetland (2,000 m²) in Urai (initial level of oil pollution of 10.5 g/l), a preliminary mechanical collection of oil was applied (75% removal) followed by a triple treatment with `Rhoder'. It resulted in an overall treatment efficiency of 94%. Relatively inferior results of bioremediation of the 10,000 m² wetland in Vyngayakha (65% removal) and the 1,000 m² marshy peat soil in Nizhnevartovsk (19% removal) can be attributed to the very high initial level of oil pollution (24.3 g/l and >750 g/g dry matter, respectively) aggravated by the fact that it was impossible to apply a preliminary mechanical collection of oil on these sites. A possible strategy for remediation of such heavily polluted sitesis discussed.     

Pressurized Pepsin Digestion in Proteomics: AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.In-depth characterization and quantitation of protein isoforms, including post-translationally modified proteins, are challenging goals of contemporary proteomics. Traditionally, top-down (1, 2) and bottom-up (3, 4) proteomics have been two distinct analytical paths for liquid-based proteomics analysis. Top-down proteomics is the mass spectrometry (MS)-based characterization of intact proteins, whereas bottom-up proteomics requires a chemical or enzymatic proteolytic digestion of all proteins into peptides prior to MS analysis. Both strategies have their own strengths and challenges and can be thought of as complementary rather than competing analytical techniques.In a top-down proteomics approach, proteins are usually separated by one- or two-dimensional liquid chromatography (LC) and identified using high performance MS (5, 6). This approach is very attractive because it allows the identification of protein isoforms arising from various amino acid modifications, genetic variants (e.g. single nucleotide polymorphisms), mRNA splice variants, and multisite modifications (7) (e.g. specific histone modifications) as well as characterization of proteolytic processing events. However, there are several challenges that have limited the broad application of the approach. Typically, intact proteins are less soluble than their peptide complement, which effectively results in greater losses during various stages of sample handling (i.e. limited sensitivity). Similarly, proteins above ∼40–50 kDa in size are more difficult to ionize, detect, and dissociate in most high throughput MS work flows. Additionally, major challenges associated with MS data interpretation and sensitivity, especially for higher molecular mass proteins (>100 kDa) and highly hydrophobic proteins (e.g. integral membrane proteins), remain largely unsolved, thus limiting the applicability of top-down proteomics on a large scale.Bottom-up proteomics approaches have broad application because peptides are easier to separate and analyze via LC coupled with tandem mass spectrometry (MS/MS), offering a basis for more comprehensive protein identification. As this method relies on protein digestion (which produces multiple peptides for each protein), the sample complexity can become exceedingly large, requiring several dimensions of chromatographic separations (e.g. strong cation exchange and/or high pH reversed phase) prior to the final LC separation (typically reversed phase (RP)¹ C₁₈), which is oftentimes directly coupled with the mass spectrometer (3, 8). In general, the bottom-up analysis rarely achieves 100% sequence coverage of the original proteins, which can result in an incorrect/incomplete assessment of protein isoforms and combinatorial PTMs. Additionally, the digested peptides are not detected with uniform efficiency, which challenges and distorts protein quantification efforts.Because the data obtained from top-down and bottom-up work flows are complementary, several attempts have been made to integrate the two strategies (9, 10). Typically, these efforts have utilized extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Results so far have encouraged us to consider on-line digestion methods for integrating top-down and bottom-up proteomics in a higher throughput fashion. Such an on-line digestion approach would not only benefit in terms of higher sample throughput and improved overall sensitivity but would also allow a better correlation between the observed intact protein and its peptide digestion products, greatly aiding data analysis and protein characterization efforts.So far, however, none of the on-line integrated methods have proven robust enough for routine high throughput analyses. One of the reasons for this limited success relates to the choice of the proteolytic enzyme used for the bottom-up segment. Trypsin is by far the most widely used enzyme for proteome analyses because it is affordable (relative to other proteases), it has been well characterized for proteome research, and it offers a nice array of detectable peptides due to a fairly even distribution of lysines and arginines across most proteins. However, protein/peptide RPLC separations (optimal at low pH) are fundamentally incompatible with on-line trypsin digestion (optimal at pH ∼ 8) (11, 12). Therefore, on-line coupling of trypsin digestion and RPLC separations is fraught with technological challenges, and proposed solutions (12) have not proven to be robust enough for integration into demanding high throughput platforms.Our approach to this challenge was to investigate alternative proteases that may be more compatible with automated on-line digestion, peptide separation, and MS detection. Pepsin, which is acid-compatible (i.e. it acts in the stomach to initially aid in the digestion of food) (13), is a particularly promising candidate. This protease has previously been successfully used for the targeted analyses of protein complexes, hydrogen/deuterium exchange experiments (14, 15), and characterization of biopharmaceuticals (16, 17). Generally, pepsin preferentially cleaves the peptide bond located on the N-terminal side of hydrophobic amino acids, such as leucine and phenylalanine, although with less specificity than the preferential cleavage observed for trypsin at arginine and lysine. The compatibility of pepsin with typical LC-MS operation makes it an ideal choice for the development of novel approaches combining protein digestion, protein/peptide separation, and MS-based protein/peptide identification.To develop an automated system capable of simultaneously capturing top-down and bottom-up data, enzyme kinetics of the chosen protease must be extremely fast (because one cannot wait hours as is typical when performing off-line proteolysis). Another requirement is the use of immobilized enzyme or a low enough concentration of the enzyme such that autolysis products do not obscure the detection of substrate peptides. The latter was a concern when using pepsin because prior hydrogen/deuterium exchange experiments used enzyme:substrate ratios up to 1:2 (18, 19). To test whether or not such a large concentration of pepsin was necessary, we performed pepsin digestion at ratios of 1:20. Many alternative energy inputs into the system were considered for speeding up the digestion. For instance, it has been shown that an input of ultrasonic energy could accelerate the reaction rate of a typical trypsin digestion while using small amounts of a protease (20). Because ultrasonic energy results in an increase of temperature and microenvironments of high pressure, it has been hypothesized that the higher temperature was the component responsible for the enhanced enzyme activity (21). López-Ferrer et al. (22, 23), however, have demonstrated that application of higher pressure with incorporation of a Barocycler alone can make trypsin display faster enzyme kinetics. This phenomenon can easily be integrated with an LC separation (which already operates at elevated pressure) to enable an automatable ultrarapid on-line digestion LC-MS proteomics platform. Herein, we refer to this platform as the fast on-line digestion system (FOLDS) (23). Although FOLDS has been described before using trypsin, here the system is characterized with pepsin, and the results obtained are compared with results attainable with trypsin. Like trypsin, pepsin produced efficient protein digestion in just a few minutes when placed under pressure. Because of the natural maximal activity of pepsin at low pH, the FOLDS can be incorporated with a RePlay (Advion Biosciences, Ithaca, NY) system, and this powerful combination is what ultimately makes the integration of top-down and bottom-up proteomics analyses possible. The integrated analysis begins with a chromatographic separation of intact proteins. The separated proteins are then split into two streams. One stream proceeds directly to the mass spectrometer for MS and/or tandem MS analysis. The second stream is split into a long capillary where the chromatographic separation of the proteins is maintained, but their arrival to the mass spectrometer for detection is delayed. This is in essence the concept of RePlay (24, 25). Herein, we have taken the RePlay a step further by implementing our FOLDS technology into the second split delayed stream of proteins. While these delayed proteins travel down the long and narrow capillary, we exposed them to pepsin where, in combination with the pressure, the proteins are quickly and reproducibly digested. These peptide fragments are subsequently subjected to MS and/or tandem MS analysis. The FOLDS RePlay system allows the rapid and robust incorporation of the integrated top-down bottom-up proteomics work flow with the ability to not only identify proteins but also to sequence multisite/combinatorial PTMs because all detected peptides (from the FOLDS analysis) are confined to the original chromatographic peak of the protein they were derived from. The analysis of protein mixtures using this integrated strategy reduces the total amount of samples required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage.     

Therapeutic potency of pharmacological adenosine receptors agonist/antagonist on cancer cell apoptosis in tumor microenvironment,current status,and perspectives

The hypoxic niche of tumor leads to a tremendous increase in the extracellular adenosine concentration through alteration of adenosine metabolism in the tumor microenvironment (TME). This consequently affects cancer progression, local immune responses, and apoptosis of tumor cells. Regulatory effect of adenosine on apoptosis in TME depends on the cancer cell type, pharmacological characteristics of adenosine receptor subtypes, and the adenosine concentration in the tumor niche. Exploiting specific pharmacological adenosine receptor agonist and antagonist inducing apoptosis in cancer cells can be considered as a proper procedure to control cancer progression. This review summarizes the regulatory role of adenosine in cancer cell apoptosis for a better understanding, and hence better management of the disease.     

The place of inactivated actin and its kinetic predecessor in actin folding-unfolding

The kinetics of actin unfolding induced by guanidine hydrochloride of different concentrations was studied. The parametric representation of the kinetic dependencies of tryptophan fluorescence intensity changes recorded at two wavelengths allowed us to detect and characterize a new essentially unfolded kinetic intermediate. Its characteristics suggested that this intermediate state is a premolten globule. It was shown that the equilibrium transition between inactivated and completely unfolded states is also a two-step process and proceeds via an essentially unfolded kinetic intermediate. The new kinetic pathway of actin unfolding--refolding was proposed. According to it, the founded essentially unfolded kinetic state is the on-pathway intermediate, while inactivated actin is the off-pathway misfolded state stabilized by aggregation of partially folded macromolecules of protein.