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PubMed1998

Solution structure of the epidermal growth factor (EGF)-like module of human complement protease C1r, an atypical member of the EGF family.

The calcium-dependent interaction between C1r and C1s, the two homologous serine proteases of the first component of human complement C1, is mediated by their N-terminal regions. The latter comprise an epidermal growth factor (EGF)-like module exhibiting the consensus sequence characteristic of Ca(2+)-binding EGF modules, surrounded by two CUB modules. Due to its Ca2+ binding ability, the C1r EGF-like module (C1r-EGF) is supposed to participate in the C1r-C1s interaction. An additional interesting feature of C1r-EGF is the unusually large loop connecting the first two conserved cysteine residues. The solution structure of synthetic C1r-EGF (residues 123-175) has been determined using nuclear magnetic resonance and combined simulated annealing-restrained molecular dynamics calculations. The resulting family of 19 structures is characterized by a well-ordered C-terminal part (residues Cys 144-Ala174) with a backbone rmsd of 0.7 A and a disordered N-terminal, including the large loop between the first two cysteines (Cys129 and Cys144). This loop is known to be surface exposed and may be expected to participate in domain-domain or protein-protein interactions. In its C-terminal part, C1r-EGF possesses the characteristic EGF fold with a major and a minor beta-sheet. The latter comprises a beta-bulge, and comparison with other EGF-like modules reveals the existence of two distinct structural and sequential motifs in the bulged part. Additional experiments in the presence of 80 mM Ca2+ did not show significant structural variation of C1r-EGF, in keeping with previous observations on blood-clotting factors IX and X.

Amino Acid Sequence↗
PubMed1998

Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides.

Acyltransferases↗
PubMed1999

Solution structure of the N-terminal F1 module pair from human fibronectin.

Multiple sites within the N-terminal domain (1-5F1) of fibronectin have been implicated previously in fibronectin matrix assembly, heparin binding, and binding to cell surface proteins of pathogenic bacteria. The solution structure of 1F1(2)F1, the N-terminal F1 module pair from human fibronectin, has been determined using NMR spectroscopy. Both modules in the pair conform to the F1 consensus fold. In 4F1(5)F1, the only other F1 module pair structure available, there is a well-defined intermodule interface; in 1F1(2)F1, however, there is no detectable interface between the modules. Comparison of the backbone 15N-{1H} NOE values for both module pairs confirms that the longer intermodule sequence in 1F1(2)F1 is flexible and that the stabilization of the 4F1 C-D loop observed in 4F1(5)F1, as a result of the intermodule interface, is not observed in 1F1(2)F1.

Amino Acid Sequence↗
PubMed2000

Synthesis and characterization of a beta-hairpin peptide that represents a 'core module' of bovine pancreatic trypsin inhibitor (BPTI).

A new strategy for the design and construction of peptide fragments that can achieve defined, nativelike secondary structure is presented. The strategy is based upon the hypothesis that 'core elements' of a protein, synthesized in a single polypeptide molecule, will favor nativelike structure, and that by incorporating a cross-link, nativelike core structure will dominate the ensemble as the more extended conformations are excluded. 'Core elements' are the elements of packed secondary structure that contain the slowest exchanging backbone amide protons in the native protein. The 'core elements' in bovine pancreatic trypsin inhibitor (BPTI) are the two long strands of antiparallel beta-sheet (residues 18-24 and 29-35) and the small beta-bridge (residues 43-44). To test the design strategy, we synthesized an 'oxidized core module', which contains the antiparallel strands connected by a modified reverse turn (A27 replaced by D), a natural disulfide cross-link at the open end of the hairpin, and N- and C-termini blocking groups. A peptide with identical sequence but lacking the disulfide cross-link at the open end was used as the 'reduced core module' control. The conformational behavior of both peptides was examined using (1)H NMR spectroscopy. Chemical shift dispersion, chemical shift deviation from random coil values, sequential and long-range NOEs, and H/D amide exchange rates were compared for the two peptides. We conclude that the ensemble of oxidized and reduced core module conformations samples both nativelike 4:4 and non-native 3:5 beta-hairpin structure, and that the oxidized module samples nativelike structure for a greater fraction of the time than the reduced module.

Amino Acid Sequence↗
PubMed1995

Effect of modulator sorption on gradient shape in ion-exchange chromatography.

Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Adsorption↗
PubMed2001

Composition dependence of the nucleation energy of iron antimonides from modulated elemental reactants.

Modulated elemental reactants containing iron and antimony were found to react at low temperature (T < 200 degrees C) forming either FeSb(2) or FeSb(3) depending on both the layer thicknesses and the overall composition of the initial reactant. For films containing 75% antimony and 25% iron, the metastable compound FeSb(3) was observed to nucleate and grow if the layer thickness was below approximately 35 A. Above this critical thickness for the modulation, annealing led to the formation of FeSb(2). This, combined with low-angle diffraction data, suggests that the initial interdiffusion between iron and antimony layers in an elementally modulated reactant results in the formation of an amorphous reaction intermediate if the layering thickness is less than 35 A. For modulated reactants with composition between 70 and 90 atomic % antimony and below this critical layer thickness, the metastable compound FeSb(3) formed. In more iron-rich modulated reactants FeSb(2) nucleates. The nucleation temperature and the nucleation energy of FeSb(3) were found to be a function of the composition of the amorphous intermediate, while those of FeSb(2) were found to be relatively independent of composition.

Journal Article↗
PubMed2003

Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase.

Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).

Anti-Bacterial Agents↗
PubMed2000

Reduction-sensitive lipopolyamines as a novel nonviral gene delivery system for modulated release of DNA with improved transgene expression.

We have designed and synthesized original cationic lipids for modulated release of DNA from cationic lipid/DNA complexes. Our rationale was that modulated degradation of the lipids during or after penetration into the cell could improve the trafficking of DNA to the nucleus resulting in increased transgene expression. The new reduction-sensitive lipopolyamines (RSL) harbor a disulfide bridge within different positions in the backbone of the lipids as biosensitive function. A useful synthetic method was developed to obtain, with very good yields and reproducibility, unsymmetrical disulfide-bridged molecules, starting from symmetrical disulfides and thiols. The new lipopolyamines are good candidates as carriers of therapeutic genes for in vivo gene delivery. To optimize the transfection efficiency in these novel series, we have carried out structure-activity relationship studies by placing the disulfide bridge at different positions in the backbone of the cationic lipid and by systematic variation of lipid chain length. Results indicate that the transfection level can be modulated as a function of the location of the disulfide bridge in the molecule. We suggest that an early release of DNA during or after penetration into the cell, probably promoted by reduction of a disulfide bridge placed between the polyamine and the lipid, implies a total loss of transfection efficiency. On the other hand, proper modulation of DNA release by inserting the disulfide bridge between one lipid chain and the rest of the molecule brings about increased transfection efficiency as compared to previously described nondegradable lipopolyamine analogues. Finally, preliminary physicochemical characterization of the complexes demonstrates that DNA release from complexes can be modulated as a function of the surrounding reducing conditions of the complexes and of the localization of the disulfide bridge within the lipopolyamine. Our results suggest that RSL is a promising new approach for gene delivery.

Animals↗
PubMed2005

Symmetry-broken reactant motion upon phase-related symmetrically modulated excitations: application to highly selective molecular sorting.

This paper introduces a separation protocol relying on affinity chromatography that exhibits unprecedented selectivities. We submit the mixture contained in the separative medium to the simultaneous action of two symmetrically modulated excitations. The first is a uniform periodic field (e.g., electric field) with zero mean value, whereas the second is the periodic modulation of a thermodynamic parameter such as the temperature. Under appropriate tuning of the modulations with the dynamics of the discriminating chemical reaction, we predict a symmetry breaking of molecular motion: the mixture components that are addressed by their rate constants exhibit an oriented motion for a particular phase relation between the modulations of the field and the thermodynamic parameter. The resulting velocity of the mixture components depends on the rate constants and on a conjugated thermodynamic value such as the standard enthalpy of the discrimination process in the case of a temperature modulation. In particular, it may be possible to separate mixture components with identical rate constants. We use the present approach to design a protocol to sort nucleic acids by their sequence.

Algorithms↗
PubMedDate not supplied

Self-organizing task modules and explicit coordinate systems in a neural network model for 3-D saccades.

The goal of this study was to train an artificial neural network to generate accurate saccades in Listing's plane and then determine how the hidden units performed the visuomotor transformation. A three-layer neural network was successfully trained, using back-prop, to take in oculocentric retinal error vectors and three-dimensional eye orientation and to generate the correct head-centric motor error vector within Listing's plane. Analysis of the hidden layer of trained networks showed that explicit representations of desired target direction and eye orientation were not employed. Instead, the hidden-layer units consistently divided themselves into four parallel modules: a dominant "vector-propagation" class (approximately 50% of units) with similar visual and motor tuning but negligible position sensitivity and three classes with specific spatial relations between position, visual, and motor tuning. Surprisingly, the vector-propagation units, and only these, formed a highly precise and consistent orthogonal coordinate system aligned with Listing's plane. Selective "lesions" confirmed that the vector-propagation module provided the main drive for saccade magnitude and direction, whereas a balance between activity in the other modules was required for the correct eye-position modulation. Thus, contrary to popular expectation, error-driven learning in itself was sufficient to produce a "neural" algorithm with discrete functional modules and explicit coordinate systems, much like those observed in the real saccade generator.

Algorithms↗
PubMed2003

Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium.

PURPOSE: The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression. METHODS: The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers. The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp. From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively. Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal. RESULTS: P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells. For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (PPD). The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion. Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp. Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression. For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity. CONCLUSIONS: The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds. The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships. A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Solution structure of a PAN module from the apicomplexan parasite Eimeria tenella.

Micronemes, specialised organelles found in all apicomplexan parasites, secrete molecules that are essential for parasite attachment and invasion of host cells. EtMIC5 is one such microneme protein that contains eleven tandemly repeating modules. These modules have homology with the PAN module superfamily. Members of this family are found in blood clotting proteins, some growth factors and some nematode proteins. This paper presents the structure of the 9th PAN module in EtMIC5, determined using high resolution NMR. The structure shows similarities to and some differences from the N-terminal module of hepatocyte growth factor (HGF), the only previous member of the PAN family with known structure.

Amino Acid Sequence↗
PubMed2000

Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrins.

The migration of keratinocytes plays an important role in the re-epithelialization of cutaneous wounds. Zinc, copper and manganese are used in vivo for their healing properties and their mechanism of action is still only partially known. Thus, they have been shown both to promote keratinocyte proliferation and to modulate integrins expression. The aim of this study was to determine if trace elements induce an increase of the migration of keratinocytes and if this effect is related to the modulation of integrins. Two independent migration assays were used to study keratinocyte migration: the scratch assay using normal human keratinocytes and the modified Boyden chamber using HaCaT cells. Inhibition studies using function-blocking antibodies directed to alpha3, alpha6, alpha(v) and beta1 subunits were performed to investigate the modulator effect of trace elements on integrin function. In this way, zinc and copper gluconates increased alpha3, alpha(v) and beta1 function whereas manganese gluconate seems mainly able to modulate the function of alpha3 and beta1. The stimulating effect of these trace elements on keratinocyte migration does not appear related to alpha6 subunit. Thus, zinc, copper and manganese enhanced keratinocyte migration and one of the mechanisms was going through a modulation of integrin functions.

3T3 Cells↗
PubMed1996

Modulation of Ca2+ channels by G-protein beta gamma subunits.

Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.

Animals↗
PubMed1998

Schedule-selective biochemical modulation of 5-fluorouracil in advanced colorectal cancer: a multicentric phase II study.

We have recently reported high clinical activity against advanced colorectal cancer of a regimen-alternating bolus FUra, modulated by methotrexate (MTX), and continuous infusion FUra, modulated by 6-s-leucovorin (6-s-LV). Considering the low toxicity of the bolus part of this regimen and our recent in vitro finding of a strong synergism between bolus FUra and natural-beta-IFN (n-beta-IFN), this cytokine was incorporated in the bolus part of our treatment programme. Fifty-six patients with untreated, advanced, measurable colorectal cancer were treated with two biweekly cycles of FUra bolus (600 mg m(-2)), modulated by MTX (24 h earlier, 200 mg m(-2)), and n-beta-IFN (3 x 10(6) IU i.m. every 12 h, starting at the time of FUra administration for four doses), alternating with a 3-week continuous infusion of FUra (200 mg m(-2) daily), modulated by 6-s-LV (20 mg m(-2) weekly bolus). After a 1-week rest, the whole cycle (8 weeks) was repeated if indicated. A total of 5 complete and 17 partial responses were obtained (response rate, 41%; 95% confidence limits, 28-55%) in 54 assessable patients. After a median follow-up time of 36 months, five patients are still alive. Overall, the median time to treatment failure was 6.4 months. The median duration of survival was 15.0 months. There was one treatment-related death after a course of MTX --> bolus FUra/n-beta-IFN and grade III-IV toxicity occurred in 18% of the patients. As the addition of n-beta-IFN results in high toxicity, whereas the efficacy seems to be similar to that of the same regimen without the cytokine, our groups are currently randomizing the original regimen, without IFN, against standard modulated bolus FUra.

Adenocarcinoma↗
PubMed2005

Micrometre-scale silicon electro-optic modulator.

Metal interconnections are expected to become the limiting factor for the performance of electronic systems as transistors continue to shrink in size. Replacing them by optical interconnections, at different levels ranging from rack-to-rack down to chip-to-chip and intra-chip interconnections, could provide the low power dissipation, low latencies and high bandwidths that are needed. The implementation of optical interconnections relies on the development of micro-optical devices that are integrated with the microelectronics on chips. Recent demonstrations of silicon low-loss waveguides, light emitters, amplifiers and lasers approach this goal, but a small silicon electro-optic modulator with a size small enough for chip-scale integration has not yet been demonstrated. Here we experimentally demonstrate a high-speed electro-optical modulator in compact silicon structures. The modulator is based on a resonant light-confining structure that enhances the sensitivity of light to small changes in refractive index of the silicon and also enables high-speed operation. The modulator is 12 micrometres in diameter, three orders of magnitude smaller than previously demonstrated. Electro-optic modulators are one of the most critical components in optoelectronic integration, and decreasing their size may enable novel chip architectures.

Journal Article↗
PubMed2005

Detection of parallel functional modules by comparative analysis of genome sequences.

Parallel functional modules are separate sets of proteins in an organism that catalyze the same or similar biochemical reactions but act on different substrates or use different cofactors. They originate by gene duplication during evolution. Parallel functional modules provide versatility and complexity to organisms, and increase cellular flexibility and robustness. We have developed a four-step approach for genome-wide discovery of parallel modules from protein functional linkages. From ten genomes, we identified 37 cellular systems that consist of parallel functional modules. This approach recovers known parallel complexes and pathways, and discovers new ones that conventional homology-based methods did not previously reveal, as illustrated by examples of peptide transporters in Escherichia coli and nitrogenases in Rhodopseudomonas palustris. The approach untangles intertwined functional linkages between parallel functional modules and expands our ability to decode protein functions from genome sequences.

Amino Acid Sequence↗
PubMed1999

Modulation of cell surface protein expression by infection with HIV-1.

The examples discussed above show the profound influence of HIV infection on expression pattern of cell surface proteins and the functional relevance thereof. Altered cell surface pattern is involved in all aspects of HIV-induced pathogenesis such as viral spreading viral adhesion and cellular apoptosis and is an important parameter for therapeutical approaches. The regulatory mechanism is not homogenous for all proteins but includes divergent effects like modulation of gene transcription and proteolytic cleavage. Modulation by viral infection might be either a direct or an indirect effect. Various viral proteins have been implicated in direct modulation like the regulatory proteins Tat, Nef and Vpu, but also the envelope proteins gp 120 and gp41. In addition, infection by HIV-1 has been shown to modulate expression of various cytokines including IL-10 and IFN-gamma. The altered expression of various surface proteins might be an indirect effect of cytokines acting on B cells, T cells and monocytic cells. By virus capture assays the presence of further proteins on viral surface was demonstrated indicating a possible function for viral life cycle. To study the modulation of expression of those additional important surface molecules by HIV and its biological function for the pathogenesis will be the aim of further studies in our laboratory.

Apoptosis↗