Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

New Δ8(14)-15-Ketosterols with an Oxygenated Side Chain

New analogues of 3β-hydroxy-5α-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3β-hydroxy-24-methyl-22,23-oxido-5α -cholest-8(14)-en-15- ones and (22RS,23ξ,24S)-24-methyl-5α-cholesta-8(14)-ene-3β, 22,23-triol-15-one] were synthesized from (22E,24S)-3β-acetoxy-24-methyl-5α-cholesta-8(14), 22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC₅₀ 0.9±0.2 and 0.7±0.2 μM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC₅₀ 4.0±0.5 μM), (22E,24S)-3β-hydroxy-24-methyl-5α-cholesta-8(14),22- dien-15-one (IC₅₀ 3.1±0.4 μM), and the 3β,22,23-triol synthesized (IC₅₀ 6.0±1.0 μM).__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 312–319.Original Russian Text Copyright © 2005 by Flegentov, Piir, Medvedeva, Tkachev, Timofeev, Misharin.     

Mechanism of stimulation by epinephrine of active transepithelial Cl transport in isolated frog cornea

Summary In the isolated frog cornea, the effects of 0.1mm epinephrine were measured on both the transepithelial and intracellular electrical parameters. Epinephrine increased the short-circuit current (I _sc) and transepithelial electrical conductance (g _t) by 176 and 96%, respectively. The effective electromotive driving force for active transepithelial Cl transport (E _Cl) was 45 mV and agrees with the value forE _Cl calculated by a different technique in the isolated rabbit corneal epithelium (Klyce, S.D., Wong, R.K.S., 1977,J. Physiol. (London) 266: 777). With respect to the tear-side bathing solution, epinephrine caused the intracellular potential difference of shortcircuited frog corneas to decrease from –54 to –50 mV (P>0.05). The fractional resistance of the apical membrane {F(R _o)=(R_o/R_o+R_i)} whereR _o andR _i represent the resistances of the apical and basolateral membranes, respectively, decreased from 0.38±0.06 to 0.23±0.03. Using these values ofF(R _o) and the cellular conductances, the calculated Cl resistances ofR _o andR _i decreased 4.3- and 2.3-fold, respectively. However, the value forE _Cl calculated from the intracellular electrical measurements (48 mV) did not appear to change since this value was in close agreement with the value forE _Cl calculated from the effects of epinephrine on the transepithelial electrical parameters. Thus, the effects of epinephrine onI _sc andg _t can be accounted for by increases in the Cl conductance of both the apical and basolateral membranes. Epinephrine caused the potential difference across the basolateral membrane to hyperpolarize by 9 mV. All of these results are consistent with the notion that the steps in transepithelial Cl transport include uphill movement into the cell across the basolateral membrane followed by downhill movement across the apical membrane into the tear-side bathing solution.     

Preliminary compositional nutrient diagnosis norms for cowpea (Vigna unguiculata (L.) Walp.) grown on desert calcareous soil

This study calculated the compositional nutrient diagnosis (CND) norms of cowpea (Vigna unguiculata (L.) Walp.), as well as identified significant nutrient interactions of this crop growing in an irrigated calcareous desert soil. Three genotypes were distributed in rows in a 2-ha field. The soil showed high heterogeneity in its chemical properties. For statistical analysis, 86 foliar composite samples from healthy plants were used. Preliminary CND norms were developed using a cumulative variance ratio function and the ² distribution function. Means and standard deviations of row-centered log ratios V_X of five nutrients (N, P, K, Ca, and Mg) and a filling value R, which included all nutrients not chemically analyzed. Preliminary CND norms are: V_N^*=0.174±0.095, V_P^*=–2.172±0.234, V_K^*=–0.007±0.267, V_Ca^*=–0.022±0.146, V_Mg^*=–1.710±0.132, and V_R5^*=3.728±0.084. These CND norms are associated with dry bean yields higher than 1.88 t ha^–1, and are associated with the following foliar concentrations: 26.2 g N kg^–1, 2.5 g P kg^–1, 22.9 g K kg^–1, 21.6 g Ca kg^–1, and 4 g Mg kg^–1. Cowpea plants growing in desert calcareous soils took up lower amounts of N, P, and K than those considered as optimum in a previous report. Six interactions were strongly indicated for cowpea through principal component analyses: positive for Ca–Mg, and negative for N–Ca, N–Mg, Ca–P, Mg–P, and K–P. Furthermore, two interactions were identified using simple correlations, negative N–P and positive K–Ca.     

Resolution of parameters in the equivalent electrical circuit of the sodium transport mechanism across toad skin

Summary In amphibian epithelia, amiloride reduces net sodium transport by hindering the entry of sodium to the active transport mechanism, that is, by increasing the series resistance (R _ser ). Theoretically, therefore, analysis of amiloride-induced changes in potential differences and short-circuit current should yield numerical estimates of all the parameters in the equivalent electrical circuit of the sodium transport mechanism.The concept has been explored by analysis of such changes in toad skins (Xenopus laevis) bathed in hypotonic sulphate Ringer's, after exposure to varying doses of amiloride, or to amphotericin, dinitrophenol or Pitressin.The estimated values ofR _ser , of the electromotive force of the sodium pump (E _Na), and of the shunt resistance (R _sh ) were independent of the dose of amiloride employed. Skins bathed in hypotonic sulphate Ringer's exhibited a progressive rise inE _Na. Amphotericin produced a fall inR _ser , while dinitrophenol caused a fall inE _Na; washout of the drugs reversed these effects. Pitressin produced a fall in bothR _ser andR _sh , with a rise inE _Na. These results are in accord with earlier suggestions regarding the site(s) of action of these agents.     

Density functional theory study on (LiNH<Subscript>2</Subscript>)<Subscript>n</Subscript> (<Emphasis Type="Italic">n</Emphasis> = 1–5) clusters

Geometrical structures and relative stabilities of (LiNH₂)_n (n = 1–5) clusters were studied using density functional theory (DFT) at the B3LYP/6-31G* and B3LYP/6-31++G* levels. The electronic structures, vibrational properties, N–H bond dissociation energies (BDE), thermodynamic properties, bond properties and ionization potentials were analyzed for the most stable isomers. The calculated results show that the Li–N and Li–Li bonds can be formed more easily than those of the Li–H or N–H bonds in the clusters, in which NH₂ is bound to the framework of Li atomic clusters with fused rings. The average binding energies for each LiNH₂ unit increase gradually from 142 kJ mol⁻¹ up to about 180 kJ mol⁻¹ with increasing n. Natural bond orbital (NBO) analysis suggests that the bonds between Li and NH₂ are of strong ionicity. Three-center–two-electron Li–N–Li bonding exists in the (LiNH₂)₂ dimer. The N–H BDE values indicate that the change in N–H BDE values from the monomer a1 to the singlet-state clusters is small. The N–H bonds in singlet state clusters are stable, while the N–H bonds in triplet clusters dissociate easily. A study of their thermodynamic properties suggests that monomer a1 forms clusters (b1, c1, d2 and e1) easily at low temperature, and clusters with fewer numbers of rings tend to transfer to ones with more rings at low temperature. E _g, E _HOMO and E _av decrease gradually, and become constant. Ring-like (LiNH₂)_3,4 clusters possess higher ionization energy (VIE) and E _g, but lower values of E _HOMO. Ring-like (LiNH₂)_3,4 clusters are more stable than other types. A comparison of structures and spectra between clusters and crystal showed that the NH₂ moiety in clusters has a structure and spectral features similar to those of the crystal.     

Single lamellar mechanics of the human lumbar anulus fibrosus

The mechanical behavior of the entire anulus fibrosus is determined essentially by the tensile properties of its lamellae, their fiber orientations, and the regional variation of these quantities. Corresponding data are rare in the literature. The paper deals with an in vitro study of single lamellar anulus lamellae and aims to determine (i) their tensile response and regional variation, and (ii) the orientation of lamellar collagen fibers and their regional variation. Fresh human body-disc-body units (L1–L2, n=11) from cadavers were cut midsagittally producing two hemidisc units. One hemidisc was used for the preparation of single lamellar anulus specimens for tensile testing, while the other one was used for the investigation of the lamellar fiber orientation. Single lamellar anulus specimens with adjacent bone fragments were isolated from four anatomical regions: superficial and deep lamellae (3.9±0.21 mm, mean ± SD, apart from the outer boundary surface of the anulus fibrosus) at ventro-lateral and dorsal positions. The specimens underwent cyclic uniaxial tensile tests at three different strain rates in 0.15 mol/l NaCl solution at 37°C, whereby the lamellar fiber direction was aligned with the load axis. For the characterization of the tensile behavior three moduli were calculated: E_low (0–0.1 MPa), E_medium (0.1–0.5 MPa) and E_high (0.5–1 MPa). Additionally, specimens were tested with the load axis transverse to the fiber direction. From the second hemidisc fiber angles with respect to the horizontal plane were determined photogrammetrically from images taken at six circumferential positions from ventral to dorsal and at three depth levels. Tensile moduli along the fiber direction were in the range of 28–78 MPa (regional mean values). Superficial lamellae have larger E_medium (p=0.017) and E_high (p=0.012) than internal lamellae, and the mean value of superficial lamellae is about three times higher than that of deep lamellae. Tensile moduli of ventro-lateral lamellae do not differ significantly from the tensile moduli of dorsal lamellae, and E_low is generally indifferent with respect to the anatomical region. Tensile moduli transverse to the fiber direction were about two orders of magnitude smaller (0.22±0.2 MPa, mean ± SD, n=5). Tensile properties are not correlated significantly with donor age. Only small viscoelastic effects were observed. The regional variation of lamellar fiber angle is described appropriately by a regression line ||=23.2+0.130× (r²=0.55, p<0.001), where is the polar angle associated with the circumferential position. The single anulus lamella may be seen as the elementary structural unit of the anulus fibrosus, and exhibits marked anisotropy and distinct regional variation of tensile properties and fiber angles. These features must be considered for appropriate physical and numerical modeling of the anulus fibrosus.     

Convenient enzymatic resolution of (R,S)‐2‐methylbutyric acid catalyzed by immobilized lipases

The application of several immobilized lipases has been explored in the enantioselective esterification of (R,S)‐2‐methylbutyric acid, an insect pheromone precursor. With the use of Candida antarctica B, using hexane as solvent, (R)‐pentyl 2‐methylbutyrate was prepared in 2 h with c 40%, ee_p 90%, and E = 35, while Thermomyces lanuginosus leads to c 18%, ee_p 91%, and E = 26. The (S)‐enantiomer was obtained by the use of Candida rugosa or Rhizopus oryzae (2‐h reaction, c 34% and 35%, ee_p 75 and 49%, and E = 10 and 4, respectively). Under optimal conditions, the effect of the solvent, the molar ratio, and the nucleophile were evaluated.     

Stem respiration and carbon dioxide efflux of young <Emphasis Type="Italic">Populus deltoides</Emphasis> trees in relation to temperature and xylem carbon dioxide concentration

Oxidative respiration is strongly temperature driven. However, in woody stems, efflux of CO₂ to the atmosphere (E _A), commonly used to estimate the rate of respiration (R _S), and stem temperature (T _st) have often been poorly correlated, which we hypothesized was due to transport of respired CO₂ in xylem sap, especially under high rates of sap flow (f _s). To test this, we measured E _A, T _st, f _s and xylem sap CO₂ concentrations ([CO₂*]) in 3-year-old Populus deltoides trees under different weather conditions (sunny and rainy days) in autumn. We also calculated R _S by mass balance as the sum of both outward and internal CO₂ fluxes and hypothesized that R _S would correlate better with T _st than E _A. We found that E _A sometimes correlated well with T _st, but not on sunny mornings and afternoons or on rainy days. When the temperature effect on E _A was accounted for, a clear positive relationship between E _A and xylem [CO₂*] was found. [CO₂*] varied diurnally and increased substantially at night and during periods of rain. Changes in [CO₂*] were related to changes in f _s but not T _st. We conclude that changes in both respiration and internal CO₂ transport altered E _A. The dominant component flux of R _S was E _A. However, on a 24-h basis, the internal transport flux represented 9–18% and 3–7% of R _S on sunny and rainy days, respectively, indicating that the contribution of stem respiration to forest C balance may be larger than previously estimated based on E _A measurements. Unexpectedly, the relationship between R _S and T _st was sometimes weak in two of the three trees. We conclude that in addition to temperature, other factors such as water deficits or substrate availability exert control on the rate of stem respiration so that simple temperature functions are not sufficient to predict stem respiration.     

Ecosystem respiration depends strongly on photosynthesis in a temperate heath

We measured net ecosystem CO₂ flux (F _n) and ecosystem respiration (R _E), and estimated gross ecosystem photosynthesis (P _g) by difference, for two years in a temperate heath ecosystem using a chamber method. The exchange rates of carbon were high and of similar magnitude as for productive forest ecosystems with a net ecosystem carbon gain during the second year of 293 ± 11 g C m⁻² year⁻¹ showing that the carbon sink strength of heather-dominated ecosystems may be considerable when C. vulgaris is in the building phase of its life cycle. The estimated gross ecosystem photosynthesis and ecosystem respiration from October to March was 22% and 30% of annual flux, respectively, suggesting that both cold-season carbon gain and loss were important in the annual carbon cycle of the ecosystem. Model fit of R _E of a classic, first-order exponential equation related to temperature (second year; R ² = 0.65) was improved when the P _g rate was incorporated into the model (second year; R ² = 0.79), suggesting that daytime R _E increased with increasing photosynthesis. Furthermore, the temperature sensitivity of R _E decreased from apparent Q ₁₀ values of 3.3 to 3.9 by the classic equation to a more realistic Q ₁₀ of 2.5 by the modified model. The model introduces R _photo, which describes the part of respiration being tightly coupled to the photosynthetic rate. It makes up 5% of the assimilated carbon dioxide flux at 0°C and 35% at 20°C implying a high sensitivity of respiration to photosynthesis during summer. The simple model provides an easily applied, non-intrusive tool for investigating seasonal trends in the relationship between ecosystem carbon sequestration and respiration.     

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume V _c, surface O _c, radius of gyration R _s, maximum dimension of the molecule L, thickness D, and cross section radius of gyration R _sq, agree within the experimental error limits. The mean values are V _c=57±3 nm³, O _c=165±10 nm², R _s=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, R _sa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D _20,w ⁰ =(5.9±0.2)·10^-7 cm²s^-1 and (6.2±0.2)·10^-7 cm²s^-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.     

Time-dependent nanomechanics of cartilage

In this study, atomic force microscopy-based dynamic oscillatory and force-relaxation indentation was employed to quantify the time-dependent nanomechanics of native (untreated) and proteoglycan (PG)-depleted cartilage disks, including indentation modulus E_ind, force-relaxation time constant τ, magnitude of dynamic complex modulus |E^∗|, phase angle δ between force and indentation depth, storage modulus E′, and loss modulus E″. At ∼2 nm dynamic deformation amplitude, |E^∗| increased significantly with frequency from 0.22 ± 0.02 MPa (1 Hz) to 0.77 ± 0.10 MPa (316 Hz), accompanied by an increase in δ (energy dissipation). At this length scale, the energy dissipation mechanisms were deconvoluted: the dynamic frequency dependence was primarily governed by the fluid-flow-induced poroelasticity, whereas the long-time force relaxation reflected flow-independent viscoelasticity. After PG depletion, the change in the frequency response of |E^∗| and δ was consistent with an increase in cartilage local hydraulic permeability. Although untreated disks showed only slight dynamic amplitude-dependent behavior, PG-depleted disks showed great amplitude-enhanced energy dissipation, possibly due to additional viscoelastic mechanisms. Hence, in addition to functioning as a primary determinant of cartilage compressive stiffness and hydraulic permeability, the presence of aggrecan minimized the amplitude dependence of |E^∗| at nanometer-scale deformation.     

Cardiorespiratory responses of the woodchuck and porcupine to CO2 and hypoxia

Summary The burrow-dwelling woodchuck (Marmota monax) (mean body wt.=4.45±1 kg) was compared to a similar-sized (5.87±1.5 kg) but arboreal rodent, the porcupine (Erithrizon dorsatum), in terms of its ventilatory and heart rate responses to hypoxia and hypercapnia, and its blood characteristics.V _T,f,T _I andT _E were measured by whole-body plethysmography in four awake individuals of each species. The woodchuck has a longerT _E/T _TOT (0.76±0.03) than the porcupine (0.61±0.03). The woodchuck had a higher threshold and significantly smaller slope to its CO₂ ventilatory response compared to the porcupine, but showed no difference in its hypoxic ventilatory response. The woodchuck P₅₀ of 27.8 was hardly different from the porcupine value of 30.7, but the Bohr factor, –0.72, was greater than the porcupine's, –0.413. The woodchuck breathing air has PaCO₂=48 (±2) torr, PaO₂=72 (±6), pHa=7.357 (±0.01); the porcupine blood gases are PaCO₂=34.6 (±2.8), PaO₂=94.9 (±5), pHa=7.419 (±0.03), suggesting a difference in PaCO₂/pH set points. The woodchuck exhibited no reduction in heart rate with hypoxia, nor did it have the low normoxic heart rate observed in other burrowing mammals.     

Spectroscopic and potentiometric characterization of cytochromes in two Sporomusa species and their expression during growth on selected substrates

The homoacetogenic bacteria Sporomusa ovata and Sporomusa sphaeroides were grown on betaine, betaine + formate, and acetoin in the absence of carbon dioxide, and the formation of membrane-bound cytochromes was determined. In S. sphaeroides, the growth substrate had little influence on the expression of cytochromes. In contrast, membranes from betaine-or acetoin-grown S. ovata cells had an 11-or 3-fold higher cytochrome b content than cells grown on betaine + formate. The cytochrome c content was reduced below the detection level after growth on the latter two substrates. The cytochromes in the membranes of S. sphaeroides and S. ovata were characterized by low-temperature difference spectroscopy, hemochrome difference spectroscopy, and redox potentiometry. Membranes of S. ovata were shown to contain two b-type cytochromes with E_m,7=-153±10 mV and E_m,7=-226±14 mV and two c-type cytochromes with E_m,7=-86±6 mV and E_m,7=-265±10 mV. In S. sphaeroides also two b-type cytochromes with E_m,7=-165±7 mV and E_m,7=-241±2 mV and two c-type cytochromes with E_m,7=-101±4 mV and E_{m, 8.5}=-338±9 mV could be distinguished. Cell extracts of S. sphaeroides were shown to contain all the enzymes of the acetyl-CoA (Wood) pathway. The degradation pathways of the substrates tested and the possible role of the cytochromes are discussed.Abbreviations E_m,7 midpoint potential at pH 7 and 25°C - H₄F tetrahydrofolate     

Ion transport across the early chick embryo: I. Electrical measurements,ionic fluxes and regional heterogeneity

The chick blastoderm at the stage of late gastrula is a flat disc formed by three cell layers and exhibiting epithelial properties. Blastoderms were cultured in miniature chambers and their electrophysiological characteristics were determined under Ussing conditions.Under open-circuit condition and identical physiological solutions on both sides, spontaneous transblastodermal potential difference (V _oc) of –7.5±3.3 mV (ventral side positive) was measured. Under short-circuit condition (transblastodermal V = 0 mV), the blastoderm generated short-circuit current (I _sc) of 21±8 A/cm², which was entirely dependent on extracellular sodium, sensitive to ouabain applied ventrally and independent of extracellular chloride. The net transblastodermal Na⁺ flux fully accounted for the measured I _sc, both under control conditions and with ouabain. The total transblastodermal resistance (R _tot) was 390±125 cm².Frequently, the V _oc, I _sc and R _tot showed spontaneous oscillations with a period of 4–5 min. Removal of endoderm and mesoderm did not significantly affect the electrical properties, indicating that the electrogenic sodium transport is generated by the ectoderm.The V _oc and I _sc measured in the area pellucida (–1.3±0.8 mV, 9.3±4.4 A/cm²) and extraembryonic area opaca (–7.8±1.1 mV, 31.2±12.7 A/cm²) were significantly different. Such a heterogeneous distribution of electrical properties can explain the presence in the blastoderm of extracellular electrical currents found by using a vibrating probe.This work was supported by the Swiss National Research Foundation (grant. 3.418-0.86 to P.K.) and by Roche Research Foundation (grant. to U.K.). We thank Drs. E. Raddatz and Y. de Ribaupierre for helpful discussions.     

Poroelasticity of cartilage at the nanoscale

Atomic-force-microscopy-based oscillatory loading was used in conjunction with finite element modeling to quantify and predict the frequency-dependent mechanical properties of the superficial zone of young bovine articular cartilage at deformation amplitudes, δ, of ∼15 nm; i.e., at macromolecular length scales. Using a spherical probe tip (R ∼ 12.5 μm), the magnitude of the dynamic complex indentation modulus, |E^∗|, and phase angle, φ, between the force and tip displacement sinusoids, were measured in the frequency range f ∼ 0.2–130 Hz at an offset indentation depth of δ₀ ∼ 3 μm. The experimentally measured |E^∗| and φ corresponded well with that predicted by a fibril-reinforced poroelastic model over a three-decade frequency range. The peak frequency of phase angle, f_peak, was observed to scale linearly with the inverse square of the contact distance between probe tip and cartilage, 1/d², as predicted by linear poroelasticity theory. The dynamic mechanical properties were observed to be independent of the deformation amplitude in the range δ = 7–50 nm. Hence, these results suggest that poroelasticity was the dominant mechanism underlying the frequency-dependent mechanical behavior observed at these nanoscale deformations. These findings enable ongoing investigations of the nanoscale progression of matrix pathology in tissue-level disease.     

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na⁺ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na⁺ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K_m= 61 ± 11 μM. Binding of Na⁺ to the empty transporter is associated with a K_m = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K_m = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na⁺ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.     

Single measurement reliability and reproducibility of volitional and magnetically-evoked indices of neuromuscular performance in adults

This study documents intra-session and inter-day reproducibility (coefficient of variation [V%]) and single measurement reliability (intra-class correlations [R_I]; standard error of a single measurement [SEM%] [95% confidence limits]) of indices of neuromuscular performance elicited during peripheral nerve magnetic stimulation. Twelve adults (five men and seven women) completed 3 assessment sessions on 3 days, during which multiple assessments of knee flexor volitional and magnetically-evoked indices of electromechanical delay (EMD_V; EMD_E), rate of force development (RFD_V; RFD_E), peak force (PF_V; P_TF_E), and compound muscle action potential latency (LAT_E) and amplitude (AMP_E) were obtained. Results showed that magnetically-evoked indices of neuromuscular performance offered statistically equivalent levels of measurement reproducibility (V%: 4.3–31.2%) and reliability (R_I: 0.98–0.51) compared to volitional indices (V%: 3.7–25.2%; R_I: 0.98–0.64), which support the efficacy of both approaches to assessment and the indices PF_V, EMD_V, EMD_E and LAT_E offer the greatest practical utility for assessing neuromuscular performance.     

Maple syrup urine disease: a possible biochemical basis for the clinical heterogeneity

Summary Nine patients with maple syrup urine disease (MSUD), of whom eight were detected by mass-screening of neonates for inherited metabolic desease, were studied to determine possible relationships between clinical features and properties of the branched-chain -keto acid dehydrogenase complex (BCKDH) in cultured lymphoblastoid cells. Based on their tolerance for leucine and on the clinical manifestations observed after 2 years of age, most could be classified into three types; classical (tolerate less than 600 mg of leucine per day, N=2), intermediate (N=3) and intermittent (N=3) types. In the other patient two of these three phenotypes were present. The BCKDH activities measured at a lower -ketoisovaleric acid concentration (0.054 mM) were 0.026±0.015 in classical, 0.118±0.016 in intermediate and 0.625±0.139 in intermittent types and 7.052±0.779 (nmol/h per milligram of protein) in two controls, respectively; the differences being statistically significant (P<0.01, classical vs intermediate types; P<0.01, intermediate vs intermittent types; P<0.01, intermittent vs control). Kinetic and immunochemical analyses of the BCKDH revealed that, although there are a few exceptions, classical, intermediate and intermittent types correspond to the enzyme properties of sigmoidal kinetics with E₁ subunit deficiency, near-sigmoidal kinetics with E₁ subunit deficiency and hyperbolic kinetics with E₂ subunit deficiency of the BCKDH, respectively.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.