An Adiabatic Calorimetry Method to Determine the Thermodynamic Characteristics of Cryoprotectants

Biophysics - Abstract—The efficiency of cryoprotectants used to protect cells from damage is usually evaluated by the changes in vital cell parameters after a freezing–thawing cycle....     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.     

Distributions of rheological parameters in populations of human erythrocytes

We have previously proposed the osmofiltration method based on a modified Hanss hemorheometer to analyze distributions of erythrocytes in their ability to pass through membrane filters with 3 microns pores. Upon decrease in medium osmolality (u) the erythrocyte volume increases. When cell volume becomes V = Vcr at u = ucr, such cell loses its ability to pass through a 3 microns pore. The flow rate of erythrocyte suspension containing cells with different ucr through a filter gradually decreases with decreasing medium osmolality. This rate becomes zero at some u = omega, when the number of non-filterable cells in the applied sample approaches the number of pores in filter. Experimental determination of the dependencies of the filtration rate on medium osmolality for various hematocrit values allows to obtain omega for each hematocrit and, thereby, to assess the distribution of erythrocytes in ucr. Here, we propose a simplified version of this method, which allows screening of the erythrocytes in heterogeneous suspensions for the distribution in ucr by measuring omega for only two hematocrit values, 0.1% and 1%. Applications of the proposed method are exemplified by analysing the erythrocyte populations of healthy donors, of patients with microspherocytosis, hemochromatosis and normal erythrocyte populations in an acidic environment.     

Fundamental step size in single cardiac and skeletal sarcomeres

In attempting to deduce the size of theelementary molecular translation step, recent experiments using singlemyosin molecules translating over actin filaments have shown aconsistent step size of 5.4 nm (10, 21). We have carriedout parallel measurements on single myofibrils from rabbit cardiacmuscle and bumblebee flight muscle. Activated specimens were releasedor stretched with a motor-imposed ramp, and the time course of lengthof individual sarcomeres was measured by projecting the image of thestriations onto a linear photodiode array and tracking the spacingbetween A-band centroids. We confirmed the 5.4-nm step. Withsubnanometer precision, however, we find that this value is two timesthat of a more fundamental step size of 2.7 nm. Step sizes were always integer multiples of 2.7 nm, whether the length change was positive ornegative. This value is equal to the linear repeat of actin monomersalong the thin filament, a result that ties dynamic events to molecularstructure and places narrow constraints on any proposed molecular mechanism.

     

Long-term circulation of vaccine-derived poliovirus that causes paralytic disease

Successful implementation of the global poliomyelitis eradication program raises the problem of vaccination against poliomyelitis in the posteradication era. One of the options under consideration envisions completely stopping worldwide the use of the Sabin vaccine. This strategy is based on the assumption that the natural circulation of attenuated strains and their derivatives is strictly limited. Here, we report the characterization of a highly evolved derivative of the Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a 7-month-old immunocompetent baby in an apparently adequately immunized population. Analysis of the genome of this isolate showed that it is a double (type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations accumulated in both the type 1-derived and type 2-derived portions of the recombinant genome suggests that both had diverged from their vaccine predecessors approximately 2 years before the onset of the illness. This fact, along with other recent observations, points to the possibility of long-term circulation of Sabin vaccine strain derivatives associated with an increase in their neurovirulence. Comparison of genomic sequences of this and other evolved vaccine-derived isolates reveals some general features of natural poliovirus evolution. They include a very high preponderance and nonrandom distribution of synonymous substitutions, conservation of secondary structures of important cis-acting elements of the genome, and an apparently adaptive character of most of the amino acid mutations, with only a few of them occurring in the antigenic determinants. Another interesting feature is a frequent occurrence of tripartite intertypic recombinants with either type 1 or type 3 homotypic genomic ends.     

Effect of Biofeedback Training of Sensorimotor and β1EEG Rhythms on Attention Parameters

The effects of the EEG–biofeedback (EEG–BFB) procedure, aimed at increasing the sensorimotor (12–15 Hz) and (15–18 Hz) rhythms on the psychological and electrophysiological parameters of attention, were studied using the methods of scalp recording of evoked potentials in the bistimulus paradigm Go/No–Go and a psychological attention test (Test of Variables of Attention; TOVA). Twenty-five children with attention disorders were included in the study. EEG–BFB sessions significantly improved the attention, behavior, and school study results in 19 (76%) children. In these cases, a significant increase in the amplitude of the inhibitory component in the frontocentral leads and improvement of the TOVA parameters were found.     

Catch-bond model derived from allostery explains force-activated bacterial adhesion

High shear enhances the adhesion of Escherichia coli bacteria binding to mannose coated surfaces via the adhesin FimH, raising the question as to whether FimH forms catch bonds that are stronger under tensile mechanical force. Here, we study the length of time that E. coli pause on mannosylated surfaces and report a double exponential decay in the duration of the pauses. This double exponential decay is unlike previous single molecule or whole cell data for other catch bonds, and indicates the existence of two distinct conformational states. We present a mathematical model, derived from the common notion of chemical allostery, which describes the lifetime of a catch bond in which mechanical force regulates the transitions between two conformational states that have different unbinding rates. The model explains these characteristics of the data: a double exponential decay, an increase in both the likelihood and lifetime of the high-binding state with shear stress, and a biphasic effect of force on detachment rates. The model parameters estimated from the data are consistent with the force-induced structural changes shown earlier in FimH. This strongly suggests that FimH forms allosteric catch bonds. The model advances our understanding of both catch bonds and the role of allostery in regulating protein activity.     

Qualitative finger and palmar dermatoglyphics: sexual dimorphism in the Chuvashian population of Russia

Qualitative finger and palmar dermatoglyphics of 547 individuals (293 males, 254 females) belonging to the Chuvashian population of Russia were studied to determine sexual dimorphism. The pattern types are not uniformly distributed on 10 fingers. Sex difference is homogeneous in all fingers whereas palmar patterns reflect the better sex variations for three palmar configurational areas (II, III, and IV). This is perhaps due to embryological development, having a relatively longer growth period compared with fingers (Cummins 1929). The present results of the Chuvashian population are not similar to the results of the five Indian populations of our previous study (Karmakar et al. 2002), perhaps due to a major ethnic difference.