Characterization of conformational deformation-coupled interaction between immunoglobulin G1 Fc glycoprotein and a low-affinity Fcγ receptor by deuteration-assisted small-angle neutron scattering

A recently developed integrative approach combining varied types of experimental data has been successfully applied to three-dimensional modelling of larger biomacromolecular complexes. Deuteration-assisted small-angle neutron scattering (SANS) plays a unique role in this approach by making it possible to observe selected components in the complex. It enables integrative modelling of biomolecular complexes based on building-block structures typically provided by X-ray crystallography. In this integrative approach, it is important to be aware of the flexible properties of the individual building blocks. Here we examine the ability of SANS to detect a subtle conformational change of a multidomain protein using the Fc portion of human immunoglobulin G (IgG) interacting with a soluble form of the low-affinity Fcγ receptor IIIb (sFcγRIIIb) as a model system. The IgG-Fc glycoprotein was subjected to SANS in the absence and presence of 75%-deuterated sFcγRIIIb, which was matched out in D₂O solution. This inverse contrast-matching technique enabled selective observation of SANS from IgG-Fc, thereby detecting its subtle structural deformation induced by the receptor binding. The SANS data were successfully interpreted by considering previously reported crystallographic data and an equilibrium between free and sFcγRIIIb-bound forms. Our SANS data thus demonstrate the applicability of SANS in the integrative approach dealing with biomacromolecular complexes composed of weakly associated building blocks with conformational plasticity.     

Synthesis of both enantiomers of 1,2,3,4-tetrahydroisoquinoline derivative IPPAM-1 and enantio-dependency of its positive allosteric modulation of prostacyclin receptor

We present a practical synthesis of both enantiomers of 1,2,3,4-tetrahydroisoquinoline derivative IPPAM-1 (1), which is a positive allosteric modulator (PAM) of prostacyclin receptor (IP) and a candidate for treatment of pulmonary arterial hypertension without the side effects caused by IP agonists. Assay of cAMP production by CHO-K1 cells stably expressing human IP clearly demonstrated that the IPPAM activity resides exclusively on the R-form of 1.     

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody–polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay as an additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA.     

Superficial aponeurosis of human gastrocnemius is elongated during contraction: implications for modeling muscle-tendon unit.

Two questions were addressed in this study: (1) how much strain of the superficial aponeurosis of the human medial gastrocnemius muscle (MG) was obtained during voluntary isometric contractions in vivo, (2) whether there existed inhomogeneity of the strain along the superficial aponeurosis. Seven male subjects, whose knees were extended and ankles were flexed at right angle, performed isometric plantar flexion while elongation of superficial aponeurosis of MG was determined from the movements of the intersections made by the superficial aponeurosis and fascicles using ultrasonography. The strain of the superficial aponeurosis at the maximum voluntary contraction, estimated from the elongation and length data, was 5.6+/-1.2%. There was no significant difference in strain between the proximal and distal parts of the superficial aponeurosis. Based on the present result and that of our previous study for the same subjects (J. Appl. Physiol 90 (2001) 1671), a model was formulated for a contracting uni-pennate muscle-tendon unit. This model, which could be applied to isometric contractions at other angles and therefore of wide use, showed that similar strain between superficial and deep aponeuroses of MG contributed to homogeneous fascicle length change within MG during contractions. These findings would contribute to clarifying the functions of the superficial aponeurosis and the effects of the superficial aponeurosis elongation on the whole muscle behavior.     

Halothane enhances acetylcholine release by decreasing dopaminergic activity in rat striatal slices

The present study investigated the effect of halothane on acetylcholine (ACh) and dopamine (DA) release from the rat striatum. Halothane decreased DA release in a concentration-dependent manner, while increased ACh release. In our previous investigation, a volatile anesthetic, halothane, inhibited DA release from the rat striatal slices in a concentration-dependent manner. Although the release of ACh from cholinergic interneurons is tonically modulated by DA in the striatum, the effect of halothane on the relationship between the release of ACh and DA has not been discussed. Using double-labeled techniques, we investigated the effect of halothane on ACh and DA release simultaneously. The slices were incubated with [14C]-choline and [3H]-DA and superfused with modified Krebs solution containing 1 microM of hemicholinium-3. We applied electrical field stimulation (2 Hz, 240 shocks), and the amount of the release of radioactivity evoked by stimulation was calculated by subtraction of the basal radioactive outflow from the total outflow at the beginning of the respective stimulation periods. The effects of drugs on the release were expressed as the ratio of stimulation-evoked fractional releases (FR), measured in the presence and absence (FRS2/FRS1) of the drug. Halothane decreased DA release in a concentration-dependent manner (FRS2/FRS1=0.767+/-0.021, 0.715+/-0.026, 0.671+/-0.014 and 0.639+/-0.033 at the concentration of 0, 0.5, 2 and 4%, respectively), while ACh release showed a biphasic change in the presence of different concentrations of halothane. The release of ACh was significantly increased at the concentration of 2%, but not at 0.5 or 4%. Halothane failed to increase the release of ACh in striatal slices after lesion by 6-OH-dopamine. The application of amphetamine reduced the release of ACh and abolished the effect of halothane. These results indicate that the effect of halothane on ACh release is indirect: it increases the release by attenuating the inhibitory effect of DA released from the nigro-striatal pathway. The nonsynaptic interaction between DA and ACh release is involved in the effect of halothane on ACh release.     

Alternate muscle activity observed between knee extensor synergists during low-level sustained contractions.

To determine quantitatively the features of alternate muscle activity between knee extensor synergists during low-level prolonged contraction, a surface electromyogram (EMG) was recorded from the rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) in 11 subjects during isometric knee extension exercise at 2.5% of maximal voluntary contraction (MVC) for 60 min (experiment 1). Furthermore, to examine the relation between alternate muscle activity and contraction levels, six of the subjects also performed sustained knee extension at 5.0, 7.5, and 10.0% of MVC (experiment 2). Alternate muscle activity among the three muscles was assessed by quantitative analysis on the basis of the rate of integrated EMG sequences. In experiment 1, the number of alternations was significantly higher between RF and either VL or VM than between VL and VM. Moreover, the frequency of alternate muscle activity increased with time. In experiment 2, alternating muscle activity was found during contractions at 2.5 and 5.0% of MVC, although not at 7.5 and 10.0% of MVC, and the number of alternations was higher at 2.5 than at 5.0% of MVC. Thus the findings of the present study demonstrated that alternate muscle activity in the quadriceps muscle 1) appears only between biarticular RF muscle and monoarticular vasti muscles (VL and VM), and its frequency of alternations progressively increases with time, and 2) emerges under sustained contraction with force production levels < or =5.0% of MVC.     

Production of interferon-β by fibroblast cells on membranes prepared by extracellular matrix proteins

The fibroblast cells from normal human skin were cultured on Langmuir-Blodgett (LB) and cast membranes prepared using extracellular matrix proteins (e.g., collagen, fibronectin, laminin and vitronectin). The cell density of the fibroblast cells cultured on the cast membranes was found to be higher than that on the cast membranes made of fibronectin, vitronectin and collagen-blended membranes. This indicates that not only the primary structure of proteins but the preparation methods of the membranes, i.e., casting and LB methods, are a strong factor affecting cell growth. The concentration and production of interferon-β per unit cell were found to be higher on the LB membranes than on the cast membranes made of the same proteins except in the case of collagen. However, the cell density on the cast membranes was higher than that on the LB membranes. These results appear to result from the suppressed growth of NB1-RGB cells on the LB membranes leading to the enhanced production of interferon-β on the LB membranes. The highest production of interferon-β per unit cell was observed for the NB1-RGB cells on the collagen-blended membranes with fibronectin and vitronectin. The collagen-blended membranes appear to offer a more natural and appropriate environment for NB1-RGB cells to produce interferon-β. This revised version was published online in September 2006 with corrections to the Cover Date.