The anionic protease inhibitor BWI-1 from buckwheat seeds. Kinetic properties and possible biological role

Kinetic characteristics and effects on the growth of filamentous fungi of one of the main anionic protease inhibitors, BWI-1, isolated from buckwheat seeds, have been studied. The inhibition constants of bovine trypsin, chymotrypsin and cathepsin G from human granulocytes with BWI-1 were found to be 1.1, 67 and 200 n M , respectively. Analysis of the amino acid sequence of BWI-1 in the vicinity of the reactive site revealed its homology to the potato proteinase inhibitor I family. It is suggested that the inability of BWI-1 to bind elastase of human granulocytes is due to the basic nature of the amino acid residue (Arg) at the P_j position in its reactive site. It was demonstrated that BWI-1 was able to suppress the germination of the spores and the growth of the mycelium of two filamentous fungi.     

Clonal Interference in the Evolution of Influenza

The seasonal influenza A virus undergoes rapid evolution to escape human immune response. Adaptive changes occur primarily in antigenic epitopes, the antibody-binding domains of the viral hemagglutinin. This process involves recurrent selective sweeps, in which clusters of simultaneous nucleotide fixations in the hemagglutinin coding sequence are observed about every 4 years. Here, we show that influenza A (H3N2) evolves by strong clonal interference. This mode of evolution is a red queen race between viral strains with different beneficial mutations. Clonal interference explains and quantifies the observed sweep pattern: we find an average of at least one strongly beneficial amino acid substitution per year, and a given selective sweep has three to four driving mutations on average. The inference of selection and clonal interference is based on frequency time series of single-nucleotide polymorphisms, which are obtained from a sample of influenza genome sequences over 39 years. Our results imply that mode and speed of influenza evolution are governed not only by positive selection within, but also by background selection outside antigenic epitopes: immune adaptation and conservation of other viral functions interfere with each other. Hence, adapting viral proteins are predicted to be particularly brittle. We conclude that a quantitative understanding of influenza’s evolutionary and epidemiological dynamics must be based on all genomic domains and functions coupled by clonal interference.     

NMR spectroscopy reveals unexpected structural variation at the protein–protein interface in MHC class I molecules

It is well established that non-uniform sampling (NUS) allows acquisition of multi-dimensional NMR spectra at a resolution that cannot be obtained with traditional uniform acquisition through the indirect dimensions. However, the impact of NUS on the signal-to-noise ratio (SNR) and sensitivity are less well documented. SNR and sensitivity are essential aspects of NMR experiments as they define the quality and extent of data that can be obtained. This is particularly important for spectroscopy with low concentration samples of biological macromolecules. There are different ways of defining the SNR depending on how to measure the noise, and the distinction between SNR and sensitivity is often not clear. While there are defined procedures for measuring sensitivity with high concentration NMR standards, such as sucrose, there is no clear or generally accepted definition of sensitivity when comparing different acquisition and processing methods for spectra of biological macromolecules with many weak signals close to the level of noise. Here we propose tools for estimating the SNR and sensitivity of NUS spectra with respect to sampling schedule and reconstruction method. We compare uniformly acquired spectra with NUS spectra obtained in the same total measuring time. The time saving obtained when only 1/k of the Nyquist grid points are sampled is used to measure k-fold more scans per increment. We show that judiciously chosen NUS schedules together with suitable reconstruction methods can yield a significant increase of the SNR within the same total measurement time. Furthermore, we propose to define the sensitivity as the probability to detect weak peaks and show that time-equivalent NUS can considerably increase this detection sensitivity. The sensitivity gain increases with the number of NUS indirect dimensions. Thus, well-chosen NUS schedules and reconstruction methods can significantly increase the information content of multidimensional NMR spectra of challenging biological macromolecules.     

Some Peculiarities of the Dynamics of the Immunoglobulin M Structure

Abstract

The isotropic mobility of separate regions of the intact molecule of immunoglobulin M (IgM) and its Fab and (Fc)₅ fragments was studied using spin-labeling of carbohydrate (2,2,6,6-tetramethyl-4-aminopiperidine-l-oxyl) and peptide (2,2,5,5-tetramethyl-3-dichloro-triazinylaminopyrrolidine-l-oxyl) moieties.

The spin-labeled oligosaccharide groups (OGs) in the Fab region are shown to have much more amplitude of anisotropic motion than those in the (Fc)₅ region. The spin label in the latter is evidently attached in the Cμ3 domain to one of its OGs which is probably stabilized by ionic contacts between terminal N-acetylneuraminic acid residue and the peptide moiety of the IgM molecule.

When the amount of the glycosidase-cleaved carbohydrate does not exceed 10–15%, most OGs affected are of the Fab region. Upon profound splitting (≥50%) the OGs of the (Fc)₅region are also affected; that results evidently in loosening the ionic contacts between the shortened OGs and the peptide moiety of IgM, and consequently in increasing mobility of the former.

The structure of the (Fc)₅ region of IgM is labile; after detaching this moiety from the intact IgM molecule, its structure is stabilized, but one of its domains (Cμ3) becomes more mobile than it is in the intact IgM molecule; at the same time the amplitude of anisotropic motion of OG bound here is decreased. In the latter case, this decrease depends on the sequence of spin-labeling and fragmentation.

The most probable cause of stabilization of the (Fc)₅ fragment is the heating of IgM solution to 56°C during fragmentation with trypsin. At this temperature the τ value for the (Fc)₅ fragment is unusually low, equaling 23 ns.

The spin-labeling in the peptide part of IgM occurs mostly in the Fab region which is a rather rigid moiety as expected.     

Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.     

Two stable electron-conformational states of photoactivated reaction center and their observation in primary donor recovery kinetics

We investigated the peculiarity of primary donor recovery kinetics in the reaction centers from the purple bacteriaRb. Sphaeroides at low levels of their cw photoactivation. A pronounced biphasity of the relaxation kinetics was found for the total light activating intensity <5×10¹² quanta·cm^-2·s^-1. The effect was attributed to strong dependence of an electron transfer rate constant for the reactionP ⁺Q _infA ^pup- Q_B P ⁺Q_AQ _infB ^sup- upon the RC conformational state controlled by the light. We showed the existence of two different electron-conformational states for the photoexcited RC. The first reveals itself at low intensity of cw photoactivation while the second becomes actual under the intensity >5×10¹² quanta · cm^-2 · s^-1.     

The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction

The synaptic growth of neurons during the development and adult life of an animal is a very dynamic and highly regulated process. During larval development in Drosophila new boutons and branches are added at the glutamatergic neuromuscular junction (NMJ) until a balance between neuronal activity and morphological structures is reached. Analysis of several Drosophila mutants suggest that bouton number and size might be regulated by separate signaling processes [Budnik, V., 1996. Synapse maturation and structural plasticity at Drosophila neuromuscular junctions. Curr. Opin. Neurobiol. 6, 858-867.]. Here we show a new role for Hangover as a negative regulator of bouton number at the NMJ. The hangover gene (hang) encodes a nuclear zinc finger protein. It has a function in neuronal plasticity mediating ethanol tolerance, a behavior that develops upon previous experience with ethanol. hang^AE10 mutants have more boutons and an extended synaptic span. Moreover, Hang expression in the motoneuron is required for the regulation of bouton number and the overall length of muscle innervation. However, the increase in bouton number does not correlate with a change in synaptic transmission, suggesting a mechanism independent from neuronal activity leads to the surplus of synaptic boutons. In contrast, we find that expression levels of the cell adhesion molecule Fasciclin II (FASII) are reduced in the hang mutant. This finding suggests that the increase in bouton number in hang mutants is caused by a reduction in FASII expression, thus, linking the regulation of nuclear gene expression with the addition of boutons at the NMJ regulated by cell adhesion molecules.     

The influence of a coastal upwelling event on chlorophyll <Emphasis Type="Italic">a</Emphasis> and nutrient dynamics in the surface layer of the Gulf of Finland,Baltic Sea

Temporal variation and distribution of chlorophyll a and nutrients concentration was evaluated on the basis of field observations in August 2006 in the Gulf of Finland. Strong easterly winds in August 2006 generated an upwelling event along the Estonian coast of the Gulf of Finland. It caused a drop of the water-surface temperature and nutrient enrichment of the upper layer. At first, the chlorophyll a declined in the area affected by the upwelled water due to the strong advective transport of the chlorophyll a rich waters towards the northern coast and due to the intensive water mixing and low seed population in the upwelling waters. After stabilization of the upwelling, nutrients from the upper mixed layer were consumed fast: there were no nitrites + nitrates left one week later, and phosphate concentration was under the detection limit 2 weeks later. The smaller phytoplankton size fraction showed faster response to the upwelled nutrients compared with the bigger size fraction, showing the increase in chlorophyll a content already during the stabilization of the upwelling. The increase in chlorophyll a concentration in >20-μm size fraction at stations influenced by upwelling was observed only after the relaxation of the upwelling and formation of stratification.