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The role of excitation-pattern cues and temporal cues in the frequency and modulation-rate discrimination of amplitude-modulated tones.

These experiments examine the influence of excitation-pattern cues and temporal-fine-structure cues on frequency difference limens (FDLs) measured as a function of duration. In the first three conditions, listeners were required to detect a change in carrier frequency from a baseline of 250 or 2000 Hz, for stimuli with half-amplitude durations ranging from 5 to 320 ms. In the "steady" condition, duration was manipulated by increasing the steady-state portion of the envelope between two 5-ms linear onset and offset ramps. This resulted in spectra and excitation patterns that broadened with decreasing duration. In the "modulated" condition, the carrier was amplitude modulated with a triangle function (period 10 ms) and duration was manipulated by varying the number of cycles of the modulator. In this case, the spectral envelope did not vary with duration, but the width of individual spectral lobes broadened with decreasing duration. The "low-peak-constant" condition was similar to the modulated condition, except that an increase in carrier frequency was accompanied by a decrease in the period of the modulator, so as to hold constant the frequency of the spectral lobe located roughly 100 Hz below the carrier frequency. In this condition, changes in carrier frequency resulted in minimal changes in excitation level on the low-frequency side of the excitation pattern, but changes on the high-frequency side were larger than for the first two conditions. Both the values of the FDLs, and their variation with frequency and duration, were similar in these three conditions. The fourth, "modulator varying," condition differed in that the carrier frequency was held constant and listeners were required to detect a change in modulator frequency. Thresholds were expressed as the change in frequency of the spectral side lobes adjacent to the carrier frequency ("equivalent" FDLs). Excitation-pattern cues in this condition were at least as large as in the first three conditions. However, equivalent FDLs were significantly higher than for the other three conditions. The higher FDLs are attributed to the lack of temporal fine-structure cues related to the carrier frequency. Overall, the results suggest that FDLs in the first three conditions were determined by temporal cues rather than by excitation-pattern cues. The increase of the FDLs with decreasing duration did not arise from increasing spectral splatter.

Auditory Perception↗

The selection of modulation rates for frequency modulated sound field stimuli.

Frequency modulated (FM) tones are used in sound field audiometry in order to avoid problems caused by standing waves. The FM stimuli to be used must be specified in terms of bandwidth (twice the frequency deviation), modulation rate and modulation waveform. This paper deals with selection of the modulation rate. Improved field uniformity obtained with FM tones relies upon the ear averaging the sound intensity as the frequency sweeps. The modulation rate must be less than about 1/3 of the frequency deviation in order to provide sufficient spectral components to ensure reasonable averaging across the frequency band. If the rate is too low threshold will be determined by the peak intensity during the sweep. For normal listeners a modulation rate of 5 Hz is just satisfactory but in the hearing impaired a higher rate is required because of their altered temporal integration characteristics. A suitable value for the modulation rate can be inferred from the literature on temporal integration and the acoustic characteristics of typical test rooms. The optimum rate increases with the stimulus centre frequency, but a constant rate of 20 Hz appears to be acceptable.

Audiometry↗

Membrane-bound forms of Ca2+-dependent protein modulator: Ca2+-dependent and independent binding of modulator protein to the particulate fraction from brain.

Ca2+-dependent binding of modulator protein to the particulate fraction was studied. The particulate fraction from one gram of rat brain bound in a Ca2+-dependent fashion 144 microgram of modulator protein, representing more than one third of the total soluble modulator protein in this tissue. The binding site was present in both the mitochondrial and microsomal fractions, the specific activity of the microsomes being the higher. The binding was reversible with a physiological concentration of Ca2+, and was temperature-dependent, and the site can be saturated with modulator protein (4.5 microgram modulator protein per mg of microsomal protein). Tryptic digestion of the membranes caused complete disappearance of the binding activity, but heat-treatment for 5 min at 70 degrees C caused only 40% loss of activity. The binding site may be a known or unknown enzyme(s), the activity of which is regulated by Ca2+ and modulator. Alternatively, this binding site may be a nonenzymic protein that regulates the concentration of free modulator protein in the cell.

3',5'-Cyclic-AMP Phosphodiesterases↗

Baculovirus-mediated expression of the epidermal growth factor-like modules of human factor IX fused to the factor XIIIa transamidation site in fibronectin. Evidence for a direct interaction between the NH2-terminal epidermal growth factor-like module of factor IXa beta and factor X.

Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.

Amino Acid Sequence↗

Substance P modulates NMDA responses and causes long-term protein synthesis-dependent modulation of the lamprey locomotor network.

Tachykinin immunoreactivity is found in a ventromedial spinal plexus in the lamprey. Neurons in this plexus project bilaterally and are thus in a position to modulate locomotor networks on both sides of the spinal cord. We have examined the effects of the tachykinin substance P on NMDA-evoked locomotor activity. Brief (10 min) application of tachykinin neuropeptides results in a prolonged concentration-dependent (>24 hr) modulation of locomotor activity, shown by the increased burst frequency and more regular burst activity. These effects are blocked by the tachykinin antagonist spantide II. There are at least two phases to the burst frequency modulation. An initial phase (approximately 2 hr) is associated with the protein kinase C-dependent potentiation of cellular responses to NMDA. The long-lasting phase (>2 hr) appears to be protein synthesis-dependent, with protein synthesis inhibitors causing the increased burst frequency to recover after washing for 2-3 hr. The modulation of the burst regularity is caused by a separate effect of tachykinins, because unlike the burst frequency modulation it does not require the modulation of NMDA receptors for its induction and is blocked by H8, an inhibitor of cAMP- and cGMP-dependent protein kinases. The effects of substance P were mimicked by the dopamine D2 receptor antagonist eticlopride. The effects of eticlopride were blocked by the tachykinin antagonist spantide II, suggesting that eticlopride may endogenously release tachykinins. Because locomotor activity in vitro corresponds to that during swimming in intact animals, we suggest that endogenously released tachykinins will result in prolonged modulation of locomotor behavior.

Animals↗

Differential modulation of the GYKI 53784-induced inhibition of AMPA currents by various AMPA-positive modulators in cerebellar Purkinje cells.

The effects of various (S)-alpha-amino-3-hydroxy-5-methyl-4-izoxazole-propionate (AMPA) receptor modulators on AMPA-induced whole-cell currents were compared in isolated rat cerebellar Purkinje cells. The positive modulators, aniracetam, cyclothiazide, 1-(1, 3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), and 1-(quinoxaline-6-ylcarbonyl)-piperidine (BDP-12), dose-dependently potentiated the steady-state component of AMPA currents. The negative modulator, (-)1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine (GYKI 53784), dose-dependently suppressed AMPA responses. Its concentration-response curve was shifted to the right in a parallel fashion by all positive modulators, indicating a competitive type of interaction. However, the relative potencies of the positive modulators were different with regard to the enhancement of AMPA responses and the reversal of GYKI 53784-induced inhibition, respectively. It is supposed that positive modulators act at multiple allosteric sites and that they interact with GYKI 53784 at only one of these sites.

Animals↗

N-terminal type I modules required for fibronectin binding to fibroblasts and to fibronectin's III1 module.

Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fiftBiomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706U.S.A. Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conh as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

Animals↗

Structural and functional roles of modules in hemoglobin. Substitution of module M4 in hemoglobin subunits.

The alpha- and beta-subunits of human hemoglobin consist of the modules M1, M2 + M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90-92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric alphaalphabeta- and betabetaalpha-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric alphaalphabeta-subunit drastically collapsed, while the chimeric betabetaalpha-subunit conserved the native globin structure (Wakasugi, K., Ishimori, K., Imai, K., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750-18756). SAXS data also suggested a partially disordered structure of the chimeric alphaalphabeta-subunit. Based on tryptophan fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp14 and Tyr125 would be induced in the chimeric alphaalphabeta-subunit, which would perturb the packing of the A- and H-helices and destabilize the globule structure. On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the betabetaalpha-subunit. The different stabilities of these module-substituted globins imply that modules would not always be stable "structural" units, and interactions between modules are crucial to construct stable globin subunits.

Circular Dichroism↗

Membrane permeation by multidrug-resistance-modulators and non-modulators: effects of hydrophobicity and electric charge.

This study was designed to test the hypothesis that lipophilic cationic drugs with only roughly similar structures mediate the reversal of multidrug-resistance (MDR) by interacting with membrane phospholipids. The permeation properties of MDR-modulators and non-modulators were studied by quantifying their ability to induce the leakage of Sulphan blue through the membrane of negatively charged unilamellar liposomes. Of the 22 compounds under investigation, only those bearing a net positive electric charge per molecule (z) > or = 0.2 induced dye leakage. All these efficient drugs are well-known MDR-modulators: calcium-channel blockers (propranolol, verapamil, diltiazem and dipyridamole), calmodulin antagonists (clomipramine and thioridazine) and antiparasitic agents (mepacrine, thioacridine derivatives and quinine). The non-modulators tested, including antineoplastic agents and steroids, did not induce any membrane permeation. The permeation process was a co-operative one (1.1 < Hill coefficient < 4.1) and the permeation doses inducing 50% dye leakage (PD50) were 1.9-11.2 mM. The permeation ability of the MDR-modulators (log(1/PD50)) increased significantly with octanol-buffer distributions per unit net electric charge ((logD)/z). The results provide evidence that a complex interplay occurs between the electric charge and the lipophilicity of the MDR-modulators when a dye leakage is induced through model membranes, and probably also when the MDR is reversed in leukaemic cells.

Anti-Inflammatory Agents↗

Corneal endothelial modulation: bFGF as direct mediator and corneal endothelium modulation factor as inducer.

PURPOSE: Previously reported from this laboratory are two distinct factors responsible for corneal endothelium modulation: basic fibroblast growth factor (bFGF) and the corneal endothelium modulation factor (CEMF) that is released by inflammatory cells. The altered phenotypes mediated by these two distinct factors--marked increase in cell proliferation, cell shape changes, and synthesis of fibrillar collagens--are identical. The current study sought to determine if bFGF is the direct mediator for corneal endothelium modulation and if CEMF plays a role in inducing bFGF production. METHODS: bFGF synthesis mediated by CEMF was analyzed by immunoblot assay; cycloheximide was used to block protein synthesis. bFGF-Specific antisense oligonucleotide primer was used to inhibit CEMF-mediated bFGF synthesis and to block further the autocrine activity of bFGF. Cell proliferation was measured by cell counting. The steady-state levels of RNA were determined by Northern blot analysis. CEMF was further purified to homogeneity by sequential electrophoresis, elution, and renaturation of protein. RESULTS: The synergistic effect of CEMF and bFGF on corneal endothelial cells was measured by their growth-promoting activity on quiescent corneal endothelial cells. There was a dose-dependent cell proliferation mediated by bFGF at any given CEMF concentration. Thus, bFGF at 10 ng/ml with CEMF at 2.5 micrograms/ml demonstrated saturable synergistic activity on endothelial cell proliferation. When the steady-state levels of collagen RNA were measured under these conditions, the untreated cells showed the doublets of 5.6 and 5.0 kb of alpha 2(I) collagen RNA. The cells treated simultaneously with bFGF and CEMF contained mostly lower transcript, compared to the significant level of upper transcript in control cells. However, there was no significant change in the level of 6.9-kb type IV collagen RNA qualitatively or quantitatively; nonetheless, the level of alpha 2(IV) collagen RNA was lowest in cells treated with bFGF plus CEMF. Neither exogenous bFGF nor CEMF caused induction of bFGF messenger RNA in corneal endothelial cells, whereas simultaneous treatment with bFGF and CEMF selectively enhanced the 4.9-kb transcript. When protein synthesis was inhibited by cycloheximide, bFGF synthesis was blocked in the presence of CEMF, leading to inhibition of corneal endothelium modulation. The effect on endothelial cell growth of bFGF antisense primer was analyzed. Antisense primer blocked by 50% the enhanced growth potential mediated by bFGF induced with CEMF. Finally, CEMF was purified to homogeneity: the purified protein is approximately 17 kD and assumes the modulating activities. CONCLUSIONS: These findings suggest that de novo synthesis of bFGF induced by CEMF is required for corneal endothelium modulation.

Animals↗

[Modulated extremely high frequency electromagnetic radiation of low intensity activates or inhibits respiratory burst in neutrophils depending on modulation frequency].

The influence of low-intensity modulated electromagnetic radiation of extremely high frequencies (EHF EMR) on synergistic reaction of calcium ionophore A23187 and phorbol ester PMA in activation of the respiratory burst of the peritoneal neutrophils of mice line NMRI was investigated. The production of reactive oxygen species by the neutrophils was estimated by luminol-dependent chemiluminescence technique. The cells were irradiated in the far field zone of the channel radiator for 20 min in the presence of A23187 and then were activated by PMA after switching off the irradiation. It was shown, that continuous EHF EMR (50 microW/cm2) inhibited quasi-resonantly the synergistic reaction. The maximum effect was about 25% at carrier frequency of 41.95 GHz. Modulated radiation with carrier frequency of 41.95 GHz and modulation frequency of 1 Hz activated the synergistic reaction, but at modulation frequencies of 0.1, 16 and 50 Hz inhibited one. At fixed modulation frequency of 1 Hz the nonlinear dependence of the effect on the carrier frequency was found. The synergistic reaction was activated in the frequency range of 41.95-42.05 GHz and was inhibited at the frequencies of 41.8-41.9 GHz. The effect was observed only at raised intracellular free calcium concentration and at calcium fluxes through plasma membrane. The obtained results prove the possibility of control over cell functioning by low-intensity modulated EHF EMR, presumably, manipulating by connected systems of enzyme reactions.

Animals↗

Chemical synthesis of human protein S thrombin-sensitive module and first epidermal growth factor module.

Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR: residues 47-75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76-116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluron ium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys85-Cys102) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys80-Cys93 and Cys104-Cys113) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys85-Cys102). CD studies revealed 54% of beta-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% beta-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway.

Amino Acid Sequence↗

Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions.

The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5'-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.

Blotting, Western↗

Temporal modulation transfer functions in the European starling (Sturnus vulgaris): I. Psychophysical modulation detection thresholds.

Temporal modulation transfer functions (TMTF) were obtained from four European starlings (Sturnus vulgaris) using a psychophysical Go/NoGo procedure combined with the method of constant stimuli. The TMTF for a continuous, broad-band noise of 55 dB SPL had a low-pass characteristic with a cut-off frequency of 123 Hz. For an 800 ms gated stimulus of the same sound-pressure level, the TMTF had the shape of a band-pass filter with the most sensitive modulation frequency at around 20 Hz. At 75 dB the band-pass shape of the TMTF was preserved, whereas at 35 dB SPL the TMTF had a low-pass characteristic. The cut-off frequency of the TMTF for continuous noise depends on which part of the spectrum carries the information on the envelope fluctuations. If only sound energy below 1 or 1.5 kHz is modulated, then the cut-off frequencies are 40 and 38 Hz, respectively. If only sound above 3 kHz carries the information on the modulation, then the cut-off frequency is 125 Hz and the shape of the TMTF is similar to that found for broadband noise. The results are discussed with respect to the coding of sinusoidal amplitude modulations by the auditory system and to different measures of time, frequency and intensity resolution in the starling.

Acoustic Stimulation↗

Three-dimensional NMR structure of the sixth ligand-binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities.

The sixth ligand-binding module of the low-density lipoprotein receptor contributes to the binding of apolipoprotein B100-containing lipoproteins. 1H NMR spectroscopy, DYANA and X-PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E-containing lipoproteins in addition to apolipoprotein B100-containing lipoproteins.

Algorithms↗

Responsive hybrid hydrogels with volume transitions modulated by a titin immunoglobulin module.

The I28 immunoglobulin (Ig)-like module of human cardiac titin, an elastic muscle protein, was used to cross-link acrylamide (AAm) copolymers into hybrid hydrogels. Cross-linking was accomplished through metal coordination bonding between terminal histidine tags (His tags) of the I28 module and metal-chelating nitrilotriacetic acid (NTA)-containing side chains on the copolymer. In solution, the beta-sheet structure of the I28 module unfolded with a transition midpoint of about 58 degrees C as the temperature was elevated. Hydrogels cross-linked with the I28 module demonstrated positive temperature responsiveness; they swelled to 3 times their initial volume at temperatures above the melting temperature of the cross-links. Positive temperature responsiveness is unusual for synthetic hydrogels. The I28 hybrid hydrogels demonstrate that cross-linking synthetic polymers with natural, well-characterized protein modules is a practical strategy for creating new materials with unique environmental responsiveness predictably determined by the mechanical properties of the protein cross-links. These new materials may be useful for controlled chemical delivery.

Acrylamides↗