Investigation of tanpura string vibrations using a two-dimensional time-domain model incorporating coupling and bridge friction

Tanpura string vibrations have been investigated previously using numerical models based on energy conserving schemes derived from a Hamiltonian description in one-dimensional form. Such time-domain models have the property that, for the lossless case, the numerical Hamiltonian (representing total energy of the system) can be proven to be constant from one time step
to the next, irrespective of any of the system parameters; in practice the Hamiltonian can be shown to be conserved within machine precision. Models of this kind can reproduce a jvari effect, which results from the bridge-string interaction. However the one-dimensional formulation has recently been shown to fail to replicate the jvaris strong dependence on the thread placement. As a first step towards simulations which accurately emulate this sensitivity to the thread placement, a twodimensional model is proposed, incorporating coupling of controllable level between the two string polarisations at the string termination opposite from the barrier. In addition, a friction force acting when the string slides across the bridge in horizontal direction is introduced, thus effecting a further damping mechanism. In this preliminary study, the string is terminated at the position of the thread. As in the one-dimensional model, an implicit scheme has to be used to solve the system, employing Newton's method to calculate the updated positions and momentums of each string segment. The two-dimensional model is proven to be energy conserving when the loss parameters are set to zero, irrespective of the coupling constant. Both frequency-dependent and independent losses are then added to the string, so that the model can be compared to analogous instruments. The influence of coupling and the bridge friction are investigated.

A comparison of two time-domain analysis procedures in the determination of VO2 kinetics by pseudorandom binary sequence exercise testing

The purpose of this study was to apply and compare two time-domain analysis procedures in the determination of oxygen uptake (VO2) kinetics in response to a pseudorandom binary sequence (PRBS) exercise test. PRBS exercise tests have typically been analysed in the frequency domain. However, the complex interpretation of frequency responses may have limited the application of this procedure in both sporting and clinical contexts, where a single time measurement would facilitate subject comparison. The relative potential of both a mean response time (MRT) and a peak cross-correlation time (PCCT) was investigated. This study was divided into two parts: a test-retest reliability study (part A), in which 10 healthy male subjects completed two identical PRBS exercise tests, and a comparison of the VO2 kinetics of 12 elite endurance runners (ER) and 12 elite sprinters (SR; part B). In part A, 95% limits of agreement were calculated for comparison between MRT and PCCT. The results of part A showed no significant difference between test and retest as assessed by MRT [mean (SD) 42.2 (4.2) s and 43.8 (6.9) s] or by PCCT [21.8 (3.7) s and 22.7 (4.5) s]. Measurement error (%) was lower for MRT in comparison with PCCT (16% and 25%, respectively). In part B of the study, the VO2 kinetics of ER were significantly faster than those of SR, as assessed by MRT [33.4 (3.4) s and 39.9 (7.1) s, respectively; P<0.01] and PCCT [20.9 (3.8) s and 24.8 (4.5) s; P < 0.05]. It is possible that either analysis procedure could provide a single test measurement Of VO2 kinetics; however, the greater reliability of the MRT data suggests that this method has more potential for development in the assessment Of VO2 kinetics by PRBS exercise testing.

The two-loop massless (lambda/4!)phi(4) model in nontranslational invariant domain

We study the (lambda/4!)phi(4) massless scalar field theory in a four-dimensional Euclidean space, where all but one of the coordinates are unbounded. We are considering Dirichlet boundary conditions in two hyperplanes, breaking the translation invariance of the system. We show how to implement the perturbative renormalization up to two-loop level of the theory. First, analyzing the full two and four-point functions at the one-loop level, we show that the bulk counterterms are sufficient to render the theory finite. Meanwhile, at the two-loop level, we must also introduce surface counterterms in the bare Lagrangian in order to make finite the full two and also four-point Schwinger functions. (c) 2006 American Institute of Physics.

Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the α-helical content increases from 60–64% to 80–90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 Å2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled α-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

N-terminal transcriptional activation domain of LZIP comprises two LxxLL motifs and the Host Cell Factor-1 binding motif

Host Cell Factor-1 (HCF-1, C1) was first identified as a cellular target for the herpes simplex virus transcriptional activator VP16. Association between HCF and VP16 leads to the assembly of a multiprotein enhancer complex that stimulates viral immediate-early gene transcription. HCF-1 is expressed in all cells and is required for progression through G1 phase of the cell cycle. In addition to VP16, HCF-1 associates with a cellular bZIP protein known as LZIP (or Luman). Both LZIP and VP16 contain a four-amino acid HCF-binding motif, recognized by the N-terminal β-propeller domain of HCF-1. Herein, we show that the N-terminal 92 amino acids of LZIP contain a potent transcriptional activation domain composed of three elements: the HCF-binding motif and two LxxLL motifs. LxxLL motifs are found in a number of transcriptional coactivators and mediate protein–protein interactions, notably recognition of the nuclear hormone receptors. LZIP is an example of a sequence-specific DNA-binding protein that uses LxxLL motifs within its activation domain to stimulate transcription. The LxxLL motifs are not required for association with the HCF-1 β-propeller and instead interact with other regions in HCF-1 or recruit additional cofactors.

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

The satellite tobacco necrosis virus RNA is uncapped and requires a 3′ translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30.

KH domain: one motif, two folds

The K homology (KH) module is a widespread RNA-binding motif that has been detected by sequence similarity searches in such proteins as heterogeneous nuclear ribonucleoprotein K (hnRNP K) and ribosomal protein S3. Analysis of spatial structures of KH domains in hnRNP K and S3 reveals that they are topologically dissimilar and thus belong to different protein folds. Thus KH motif proteins provide a rare example of protein domains that share significant sequence similarity in the motif regions but possess globally distinct structures. The two distinct topologies might have arisen from an ancestral KH motif protein by N- and C-terminal extensions, or one of the existing topologies may have evolved from the other by extension, displacement and deletion. C-terminal extension (deletion) requires β-sheet rearrangement through the insertion (removal) of a β-strand in a manner similar to that observed in serine protease inhibitors serpins. Current analysis offers a new look on how proteins can change fold in the course of evolution.

RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.

Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.