Single-molecule pulling experiments: when the stiffness of the pulling device matters

Using Langevin modeling, we investigate the role of the experimental setup on the unbinding forces measured in single-molecule pulling experiments. We demonstrate that the stiffness of the pulling device, K_eff, may influence the unbinding forces through its effect on the barrier heights for both unbinding and rebinding processes. Under realistic conditions the effect of K_eff on the rebinding barrier is shown to play the most important role. This results in a significant increase of the mean unbinding force with the stiffness for a given loading rate. Thus, in contrast to the phenomenological Bell model, we find that the loading rate (the multiplicative value K_effV, V being the pulling velocity) is not the only control parameter that determines the mean unbinding force. If interested in intrinsic properties of a molecular system, we recommend probing the system in the parameter range corresponding to a weak spring and relatively high loading rates where rebinding is negligible.     

Segmental flexibility in pig immunoglobulin G studied by neutron spin-echo technique

The dynamic behavior of pig immunoglobulin G in deuterium oxide solutions was investigated by the neutron spin-echo technique. This novel technique makes it possible to study intramolecular motion without introducing probes into the macromolecule. Using neutron spin-echo, the effective diffusion coefficient, D_eff, was obtained as a function of the transferred momentum, κ. For interpretation of the experimental data, two models were designed, and computed D_eff values were compared with experimental data. The rigid T-shape model was compatible with the experimental data only by assuming an unrealistically high rotational diffusion coefficient, and it was therefore unacceptable. Reasonable agreement with all available experimental data was obtained with a flexible model of immunoglobulin G molecule, which the Fab arms are assumed to wobble around the hinge region within an angle of 50°.     

Numerical modeling of molecular-motor-assisted transport of adenoviral vectors in a spherical cell

Viral gene delivery in a spherical cell is investigated numerically. The model of intracellular trafficking of adenoviruses is based on molecular-motor-assisted transport equations suggested by Smith and Simmons. These equations are presented in spherical coordinates and extended by accounting for the random component of motion of viral particles bound to filaments. This random component is associated with the stochastic nature of molecular motors responsible for locomotion of viral particles bound to filaments. The equations are solved numerically to simulate viral transport between the cell membrane and cell nucleus during initial stages of viral infection.     

Histological development of the long‐snouted seahorse Hippocampus guttulatus during ontogeny

The objective of the present study was to describe histological development of the European long‐snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the male's pouch. Digestive tract specialization occurred at 89 effective day‐degrees (D°_eff), corresponding to 15 days post partum (dpp), with development of the first intestinal loop and mucosal folding. At 118 D°_eff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage. The fovea emerged at 177 D°_eff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 D°_eff; 0–60 dpp), but the first oogonia were present at 30 D°_eff (5 dpp). In 354 D°_eff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 D°_eff. The present study demonstrates that approximately 89 and 177 D°_eff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1°C and an age of 1 month (177 D°_eff), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming mature at the age of 2 months (354 D°_eff).     

Construction of highly-effective symbiotic bacteria: Evolutionary models and genetic approaches

Using the example of N₂-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve: (i) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; (ii) the loss of genes encoding for functions that are required for autonomous performance but interfere with symbiotic functions (negative regulators of symbiosis). Therefore, the construction of effective rhizobia strains should involve improvement of sym genes activities (for instance, nif, fix, and dct genes encoding for nitrogenase synthesis or for the energy supply of N₂ fixation), as well as the inactivation of negative regulators of symbiosis identified in our lab (eff genes encoding for the transport of sugars and the production of polysaccharides and storage compounds, as well as for oxidative-reductive processes).     

Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria

Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca²⁺ channels (Ca_V3.1, Ca_V3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Ca_v3.1 and Ca_V3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca²⁺ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both Ca_V3.1 and Ca_V3.2 in co-immunoprecipitation experiments. T-type Ca²⁺ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify Ca_V3.1 and Ca_V3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction.     

Temperature and field dependent 1H-NMR relaxation data as probes of prostaglandin motional parameters in aqueous media

Proton resonance correlation times (τ_eff) for PGF₂α and a more rigid analog have been derived from the field-strength dependence of spinlattice relaxation times (T_1D) using 200 and 500 MHz observation. Those hydrogens showing τ_eff less than the value calculated for whole molecule tumbling (which applies for H-5 → H-15) also show a significantly greater temperature dependence for T_1D at 500 MHz. Minor wagging may occur at the C-7 and C-10 methylenes, and gradually increasing segmental motion is observed toward both side chain termini. A current model for the aqueous geometry of PGF₂α is developed from this data and studies of relaxation rate changes upon specific deuteration.     

Control and evaluation of high-frequency jet ventilation: mechanical lung model

Ventilation systems that operate at high-frequency and deliver small volumes have the potential to provide adequate alveolar ventilation without excessive pulmonary pressures. One way of producing high-frequency ventilation is by use of jet bursts of an input gas through a cannula controlled by a solenoid valve. This high-frequency jet ventilation has yet to be quantitatively analysed for optimal clinical use. From an analysis of the jet-producing device, we obtained a quantitative relationship which allowed us to predict the gas volume of a jet burst (V_jet) from the driving pressure (P_d), and the jet duration (t_I). The device was applied to a mechanical lung model (a tube attached to an elastic bag corresponding to the lung airway and alveolar space). We examined how the control variables of the jet ventilation system changed the bag (alveolar) volume with respect to V_jet, the volume of entrained gas, and the volume of shunted gas. Using a nitrogen washout analysis, we evaluated the operating lung volume, effective dead-space volume (V_eds), and effective ventilation rate (V_eff). We found that V_eds is independent of the individual effects of jet cycle frequency, duty cycle, cannula diameter, and entrainment fraction. While V_eds was not affected significantly by the shape of the airway, it did depend on the distance of the jet cannula tip to the ventilated bag (or alveolar region) and on the tidal volume.     

The use of gel chromatography to study rapid equilibria of the type A + B forms and is formed from C + D

A frontal gel chromatographic procedure is illustrated whereby the equilibrium constant for hybridization equilibria (A + B ? C + D) may be obtained provided the relative elution volume situation V_A = V_C <V_B = V_D may be realized. The electron transfer between reduced cytochrome c and ferricyanide is used as a model interaction, with Sephadex G-25 as the gel medium.     

Good Cascade Impactor Practice (GCIP) and Considerations for “In-Use” Specifications

The multi-stage cascade impactor (CI) is widely used to determine aerodynamic particle size distributions (APSDs) of orally inhaled products. Its size-fractionating capability depends primarily on the size of nozzles of each stage. Good Cascade Impactor Practice (GCIP) requires that these critical dimensions are linked to the accuracy of the APSD measurement based on the aerodynamic diameter size scale. Effective diameter (D _eff) is the critical dimension describing any nozzle array, as it is directly related to stage cut-point size (d ₅₀). d ₅₀ can in turn be determined by calibration using particles of known aerodynamic diameter, providing traceability to the international length standard. Movements in D _eff within manufacturer tolerances for compendial CIs result in the worst case in shifts in d ₅₀ of <±10%. Stage mensuration therefore provides satisfactory control of measurement accuracy. The accurate relationship of D _eff to d ₅₀ requires the CI system to be leak-free, which can be checked by sealing the apparatus at the entry to the induction port and isolating it from the vacuum source and measuring the rate of pressure rise before each use. Mensuration takes place on an infrequent basis compared with the typical interval between individual APSD determinations. Measurement of stage flow resistance (pressure drop; ΔP _stage) could enable the user to know that the CI stages are fit for use before every APSD measurement, by yielding an accurate measure of D _eff. However, more data are needed to assess the effects of wear and blockage before this approach can be advocated as part of GCIP.     

Evidence for Ubiquitin-Regulated Nuclear and Subnuclear Trafficking among Paramyxovirinae Matrix Proteins

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLS_bp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLS_bp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLS_bp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLS_bp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLS_bp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLS_bp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype.     

Complementary role of surface hydrolysis and intact transport in the intestinal assimilation of di- and tripeptides

The small intestine is known to possess mechanisms for intact transport and membrane hydrolysis of oligopeptides. To determine the relative role of these processes in peptide assimilation the fate of two model peptides known to be high-affinity substrates for the brush border aminooligopeptidase were studied in rat small intestine in vivo. Both 20 mM Gly-L-Pro, a potent inhibitor of peptide transport, and specific inhibitors of the aminopeptidase, 10 mM L-Ala-β-naphthylamide or the phthalimido derivative of 0.1 mM L-leucine bromomethyl ketone, reduced assimilation of L-Leu-Gly-Gly and L-Leu-L-Leu. Further inhibition was found when both transport and peptidase inhibitors were included in the intestinal perfusate suggesting that the model di- and tripeptides utilize both intact transport and surface hydrolysis for their assimilation. Although comparative kinetic parameters of intact transport (K_m = 22 mM; V = 1.9 · 10^?3μmol · s^?1 · cm^?2) and surface hydrolysis (K_m = 8.7; V = 1.1 · 10^?3) for l-Leu-l-Leu differed markedly, the relationship of peptide concentration to assimilation rate was nearly identical for intact transport and surface hydrolysis in the physiological range of 1–10 mM substrate. Both intact peptide transport and surface hydrolysis appear to be efficient and complementary processes that promote efficient assimilation of dipeptides and tripeptides. The relative importance of each assimilation process appears to depend upon the amino acid composition of the peptide nutrient.     

A simple model of the relationship between asymmetry and developmental stability

The relationship between developmental stability and morphological asymmetry is derived under the standard view that structures on each side of an individual develop independently and are normally distributed. I use developmental variance of sizes of parts, V_D, as the converse of developmental stability, and assume that V_D follows a gamma distribution. Repeatability of asymmetry, a measure of how informative asymmetry is about V_D, is quite insensitive to the variance in V_D, for example only reaching 20% when the coefficient of variation of V_D is 100%. The coefficient of variation of asymmetry, CV_FA, also increases very slowly with increasing population variation in V_D. CV_FA values from empirical data are sometimes over 100%, implying that developmental stability is sometimes more variable than any previously studied type of trait. This result suggests that alternatives to this model may be needed.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

D5 Dopamine Receptor Knockout Mice and Hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D₁, D₂, D₃, D₄, and D₅) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D₁-like receptors (D₁ and D₅) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D₁-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D₁-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D₁ receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D₅ receptor is also important in blood pressure regulation. Disruption of the D₅ or the D₁ receptor gene in mice increases blood pressure. However, unlike the D₁ receptor, the hypertension in D₅ receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V₁ vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.     

Gas transport during high-frequency ventilation: the significance of direct wash-out

Gas transport during high-frequency oscillation was investigated in vitro using CO₂ elimination from the lung surrogate as a measure of gas transport efficiency. The length of the connecting tube between the piston pump and the three-port connector did not affect gas transport efficiency if the oscillatory volume (V_DEL) was constant; inserting an additional tube between the three-port connector and the endotracheal tube decreased gas transport efficiency dramatically. In contradistinction, increasing V_DEL caused a steep rise in gas transport efficiency as soon as V_DEL surpassed the volume of the tubes connecting the lung surrogate with its surroundings. As gas transport effiency was found to be very sensitive to the net oscillatory volume, i.e. V_DEL minus the volume of the tubes connecting the lung and the surroundings, direct wash-out was considered to be an effective gas transport mechanism during high frequency oscillation. Two preliminary experiments on dogs allowed us to substantiate this hypothesis in vivo.     

Photon buildup factors in some dosimetric materials for heterogeneous radiation sources

Effective photon energy absorption (EABF_eff) and exposure buildup factors (EBF_eff) have been calculated based on the effective energy concept, for some dosimetric materials such as water, polymethyl methacrylate (PMMA), polystyrene, solid water (WT1), RW3 (Goettingen Water 3), and ABS (acrylonitrile butadiene styrene), for MV X-rays and ⁶⁰Co gamma rays. Firstly, the equivalent atomic numbers (Z _eq) of the given materials have been determined using the effective photon energies (E _eff). Then, the five-parameter geometric progression (G-P) fitting approximation has been used to calculate both EABF_eff and EBF_eff values. Since the G-P fitting parameters are not available for the E _eff values of the given materials, a linear interpolation in which a function of the logarithm of the variable is used has been performed, in order to calculate the parameters in each E _eff, which will be further used for the determination of EABF_eff and EBF_eff. In the present paper, water equivalence properties of the given materials are also discussed based on the effective buildup factors. In this study, special emphasis is placed on the calculation of EABF_eff and EBF_eff values of different materials for photons that are not monoenergetic but heterogeneous in energy, to obtain an initial and prior knowledge of the probable energy and buildup of photons at locations of interest, i.e., to understand whether the real absorbed dose occurs at the surface or somewhere inside the medium of interest.