Constitutive Monomers Research Articles

Structural diversity and chemical logic underlying the assembly of monoterpene indole alkaloids oligomers.

Covering: up to 2024This review aims to draw a parallel between all known oligomers of monoterpene indole alkaloids (MIAs) by illustrating the chemical logic underlying their assembly. For this purpose, oligomeric MIAs were first comprehensively listed and organized according to the names of the backbones of their constitutive monomers and the binding sites. From this extensive list, an oligomer network was generated and unprecedented MIA statistics were mined and shared herein. Subsequently, oligomeric MIAs were categorized according to the number of connections instigated between their monomeric components (single, double, triple, and mixed tethering), then subdivided according to the uniqueness or combination of oligomerization assembly reactions. This effort outlined oligomerization trends in a scaffold-specific manner, and established binding reactivity patterns facilitating the comprehension of the associated biosynthetic processes. At last, this review illustrates a unique initiative in crafting a comprehensive repository of machine-readable metadata for MIA oligomers that could be leveraged for chemoinformatic purposes.

Composition-Property Relationships of pH-Responsive Poly[(2-vinylpyridine)-co-(butyl methacrylate)] Copolymers for Reverse Enteric Coatings.

The taste-masking of bitter-tasting active pharmaceutical ingredients is key to ensuring patient compliance when producing oral pharmaceutical formulations. This is generally achieved via the incorporation of pH-responsive, reverse enteric polymers, that prevent the dissolution of the formulation in the oral environment, but rapidly mediate it within the gastric environment. Reverse enteric polymers are commonly applied as coatings on oral dosage forms via spray atomisation (e.g., fluidised-bed spray coating), and generally exhibit the most efficient taste-masking. However, currently used reverse enteric coatings require high mass gains (% w/w) during coating to mediate taste-masking, and thereby exhibit delayed release within the gastric environment. Therefore, there remains a need for the development of new reverse enteric coatings, that can efficiently taste-mask at low mass gains and maintain rapid release characteristics within the gastric environment. Herein we report the synthesis and evaluation of a series of addition copolymers of 2-vinylpyridine and butyl methacrylate, methyl methacrylate and isobornyl methacrylate. The thermal, solubility, and water absorption properties of the copolymers were effectively tuned by altering the mol% fraction of the constitutive monomers. Based on their physical properties, selected copolymers were preliminarily evaluated for their compatibility with fluidised-bed spray coating, and effectiveness as taste-masking reverse enteric coatings. The copolymers poly[(2-vinylpyridine)-co-(butyl methacrylate)] (mol% ratio 40:60) and poly[(2-vinylpyridine)-co-(butyl methacrylate)-co-(methyl methacrylate)] (mol% ratio 40:50:10) were found to exhibit excellent taste-masking properties following fluidised-bed spray coating onto Suglets® sugar spheres. Suglets® bearing a film coating of either copolymer (5.2-6.5% w/w mass gain) were found to effectively impede the release of a model drug formulation for up to 72 h in a simulated salivary environment, and rapidly release it (<10 min) within a simulated gastric environment. The results demonstrated the potential of poly[(2-vinylpyridine)-co-(butyl methacrylate)] copolymers to form effectively taste-masked, reverse enteric dosage forms, and suggested that these copolymers may provide improved performance compared to currently available polymers.

Classifying soft self-assembled materials via unsupervised machine learning of defects

Unlike molecular crystals, soft self-assembled fibers, micelles, vesicles, etc., exhibit a certain order in the arrangement of their constitutive monomers but also high structural dynamicity and variability. Defects and disordered local domains that continuously form-and-repair in their structures impart to such materials unique adaptive and dynamical properties, which make them, e.g., capable to communicate with each other. However, objective criteria to compare such complex dynamical features and to classify soft supramolecular materials are non-trivial to attain. Here we show a data-driven workflow allowing us to achieve this goal. Building on unsupervised clustering of Smooth Overlap of Atomic Position (SOAP) data obtained from equilibrium molecular dynamics simulations, we can compare a variety of soft supramolecular assemblies via a robust SOAP metric. This provides us with a data-driven “defectometer” to classify different types of supramolecular materials based on the structural dynamics of the ordered/disordered local molecular environments that statistically emerge within them.

Macrocephatriolides A and B: Two Guaianolide Trimers from Ainsliaea macrocephala as PTP1B Inhibitors and Insulin Sensitizers.

Macrocephatriolides A and B (1 and 2), two novel guaiane-type sesquiterpene lactone trimers possessing unique linkage patterns, were identified from the whole plant of Ainsliaea macrocephala. The trimeric architecture of 1 features a cyclohexene linkage and a methylene bridge, which were presumably constructed from three constitutive monomers via a Diels-Alder cycloaddition and a Michael addition, respectively. The three monomers of 2 were tethered by a 1,2-ethanediyl and a methylene linkage at the same time. Their complex structures were established by extensive analysis of spectroscopic data inclusive of band-selective CT-HSQC and CT-HMBC and time-dependent density functional theory (TDDFT) ECD calculations. Compound 2 showed potent inhibition against protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 26.26 ± 0.88 μM but not compound 1. In the kinetic study, compound 2 was disclosed as a competitive inhibitor of PTP1B with a Ki value of 16.34 ± 4.72 μM. In insulin-stimulated C2C12 myotubes, compound 2 dose-dependently enhanced glucose uptake by activating the insulin signaling pathway. Compound 2 might represent a new scaffold of insulin sensitizers.

High performance liquid chromatographic method with post-column detection for quantification of reducing sugars in foods

A novel liquid chromatographic analysis method with post-column detection for sugars was developed to improve existing methods in regard to operation time, selectivity, and sensitivity. This method involves separation of reducing sugars on HPLC column at 30 °C and 0.8 mL min−1 flow rate, post−column reaction of sugars with Cu(II)-neocuproine (Nc) reagent at 80 °C and 0.3 mL min−1 flow rate, and measurement of Cu(I)-Nc product at 450 nm. The proposed assay was applied to glucose, fructose, maltose, and lactose as reducing sugars. Non-reducing sucrose was determined indirectly, after conversion to its constitutive monomers glucose and fructose by hydrolysis, and analysis with a relative error from -2.41 to 2.09%. Honey, apple juice, and milk samples were evaluated as commercial products. The results obtained with the proposed assay compared to those of the alkaline Cu(II)−Nc reference method were found close to each other, and compatible with the label values of commercial products. The accuracy of the developed method was performed by spiking glucose to honey and lactose to milk samples using two different concentrations. The obtained recoveries with respect to the post-column HPLC method were between 97 and 105% for honey and 96–107% for milk. The method gave linear responses against sugar concentration with correlation coefficients greater than 0.996 for the four analytes (glucose, fructose, maltose and lactose) in a range of 9.0 - 342.3 mg L−1 with LOD values ≤ 7.4 mg L−1. With the developed method, it was possible to sensitively determine reducing sugars in various food samples at a lower temperature of post-column reaction (compared to literature values) with easy application of low cost reagents requiring minimal preliminary operation.

Controlling Exchange Pathways in Dynamic Supramolecular Polymers by Controlling Defects.

Supramolecular fibers composed of monomers that self-assemble directionally via noncovalent interactions are ubiquitous in nature, and of great interest in chemistry. In these structures, the constitutive monomers continuously exchange in-and-out the assembly according to a well-defined supramolecular equilibrium. However, unraveling the exchange pathways and their molecular determinants constitutes a nontrivial challenge. Here, we combine coarse-grained modeling, enhanced sampling, and machine learning to investigate the key factors controlling the monomer exchange pathways in synthetic supramolecular polymers having an intrinsic dynamic behavior. We demonstrate how the competition of directional vs. nondirectional interactions between the monomers controls the creation/annihilation of defects in the supramolecular polymers, from where monomers exchange proceeds. This competition determines the exchange pathway, dictating whether a fiber statistically swaps monomers from the tips or from all along its length. Finally, thanks to their generality, our models allow the investigation of molecular approaches to control the exchange pathways in these dynamic assemblies.

Programmed and Sequential Disassembly of Multi-responsive Supramolecular Protein Nanoassemblies: A Detailed Mechanistic Investigation.

The rational design of a multi-responsive protein-based supramolecular system that can predictably respond to more than one stimulus remains an essential but highly challenging goal in biomolecular engineering. Herein, we report a novel chemical method for the construction of multi-responsive supramolecular nanoassemblies using custom-designed facially amphiphilic monodisperse protein-dendron bioconjugates. The macromolecular synthons contain a globular hydrophilic protein domain site-specifically conjugated to photo-responsive hydrophobic benzyl-ether dendrons of different generations through oligo(ethylene glycol) linkers of defined length. The size of the protein nanoassemblies can be systematically tuned by choosing an appropriate dendron or linker of defined length. Exposure of protein nanoassemblies to light results in partial rather than complete disassembly of the complex. The newly formed protein nanoparticle no longer responds to light but could be disassembled into constitutive monomers under acidic conditions or by further treatment with a small molecule. More interestingly, the distribution ratio of the assembled versus disassembled states of protein nanoassemblies after photochemical reaction does not depend on dendron generation, the nature of the linker functionality or the identity of the protein, but is heavily influenced by the linker length. In sum, this work discloses a new chemical method for the rational design of a monodisperse multi-responsive protein-based supramolecular system with exquisite control over the disassembly process.

Self-Sorted, Random, and Block Supramolecular Copolymers via Sequence Controlled, Multicomponent Self-Assembly.

Multicomponent supramolecular copolymerization promises to construct complex nanostructures with emergent properties. However, even with two monomeric components, various possible outcomes such as self-sorted supramolecular homopolymers, a random (statistical) supramolecular copolymer, an alternate supramolecular copolymer, or a complex supramolecular block copolymer can occur, determined by their intermolecular interactions and monomer exchange dynamics and hence structural prediction is extremely challenging. Herein, we target this challenge and demonstrate unprecedented two-component sequence controlled supramolecular copolymerization by manipulating thermodynamic and kinetic routes in the pathway complexity of self-assembly of the constitutive monomers. Extensive molecular dynamics simulations provided useful mechanistic insights into the monomer exchange rates and free energy of interactions between the monomers that dictate the self-assembly pathway and sequence. The fluorescent nature of core-substituted naphthalene diimide monomers has been further utilized to characterize the three sequences via Structured Illumination Microscopy (SIM).

The use of extracted-ion chromatograms to quantify the composition of condensed tannin subunits.

RationaleThe analysis of tannins is currently usually done by liquid chromatography after the chemical depolymerization of tannins. However, one of the main limitations of this method is the difficulty in specifically detecting each constitutive tannin monomer in a complex matrix, as numerous compounds co‐elute with the monomers, thereby compromising the analysis.MethodsThe depolymerization of tannins under acidic conditions and in the presence of thioglycolic acid releases the various constitutive monomers, either as terminal units or as extension units. The tannin subunits were then quantified using extracted‐ion chromatogram (EIC) mass spectrometry, which required the determination of the ionization efficiency of the monomers. Thus, we used AgBF4‐assisted coupling for the hemisynthesis of the noncommercial epicatechin gallate dimer.ResultsThe EIC showed that the derivatives of the extension units were twice as ionized as the terminal units. Unlike the UV chromatograms, this new EIC‐based method is more specific and accurate because it is not impacted by the co‐elution phenomenon. This result, when added to the linearity obtained on a large range of the calibration curves, allowed for quantification of tannin subunits from EIC in complex mixtures with less pretreatment of the samples.ConclusionsDue to its specificity and sensitivity, this EIC‐based method represents a significant step toward improving the quantification of the tannin composition of samples. The results of this study should allow the oxidation markers to be quantified more accurately and taken into account, thereby providing a better indication of the actual tannin composition.

Predicted microbial secretomes and their target substrates in marine sediment.

Scientific drilling has identified a biosphere in marine sediments 1 , which contain many uncultivated microbial groups known only by their DNA sequences 2-4 . Recycling of organic matter in sediments is an important component of biogeochemical cycles because marine sediments are critical forlong-term carbon storage 5 . Turnover of carbon is hypothesized to be driven by the secretion of enzymes by microbial organisms 5-7 , which act to break down macromolecules into constitutive monomers that can be transported into cells. As such, the nature of the microbial secretome often influences the function of a community 6 . However, the microbial groups involved in this process and the biochemistry theyencode is poorly understood. Here, we show that expressed genes from 5 to 159 meters below the seafloor 8 (mbsf) encode numerous candidate peptidases and carbohydrate-active enzymes ('CAZymes') 9 targeted for secretion. The majority (90-99%) were assigned to Bacteria, of which 12% shared the highest sequence similarity with candidate phyla 10,11 . The remaining putatively secreted proteins shared highest sequence similarity with archaeal and fungal enzymes, which peak in two redox transition zones 12 . In the shallower redox zone at 30 mbsf, 20% of the transcripts encoding putative secreted peptidases were assigned to lineages 7,13,14 of uncultivated Archaea. The target compounds of the predicted secreted proteome show a preference for necromass in the form of microbial cell envelopes as well as plankton and algal detritus. The predicted fungal secreted proteome encodes CAZymes not present in the predicted bacterial or archaeal secreted proteomes, indicating that fungi putatively play a minimal but specialized role in subseafloor carbohydrate recycling.

Chronophin Dimerization Is Required for Proper Positioning of Its Substrate Specificity Loop

Mammalian phosphatases of the haloacid dehalogenase (HAD) superfamily have emerged as important regulators of physiology and disease. Many of these enzymes are stable homodimers; however, the role of their dimerization is largely unknown. Here, we explore the function of the obligatory homodimerization of chronophin, a mammalian HAD phosphatase known to dephosphorylate pyridoxal 5'-phosphate (PLP) and serine/threonine-phosphorylated proteins. The exchange of two residues in the murine chronophin homodimerization interface (chronophin(A194K,A195K)) yields a constitutive monomer both in vitro and in cells. The catalytic activity of monomeric chronophin toward PLP is strongly impaired. X-ray crystallographic studies of chronophin(A194K,A195K) revealed that dimer formation is essential for an intermolecular arginine-arginine-tryptophan stacking interaction that positions a critical histidine residue in the substrate specificity loop of chronophin for PLP coordination. Analysis of all available crystal structures of HAD hydrolases that are grouped together with chronophin in the C2a-type structural subfamily uncovered a highly conserved mode of dimerization that results in intermolecular contacts involving the substrate specificity loop. Our results explain how the dimerization of HAD hydrolases contributes to their catalytic efficiency and substrate specificity.

Abstract C42: Mitochondrial glutaminase regulation and small molecule inhibition of glutamine metabolism

Abstract The mitochondrial enzyme glutaminase isoform C (GAC) has recently gained significant attention for its critical role in supporting glutamine dependent proliferation in a variety of cancer cells and has been a significant focus for therapeutic intervention (1,2). To this end, we have combined biophysical techniques with cellular metabolic assays to gain insight as to the mechanism of action of the novel GAC inhibitor, compound 968, shown previously to inhibit Dbl (for Diffuse B-cell Lymphoma) oncogene dependent transformation (2). We have developed a fluorescence resonance energy transfer (FRET) approach, along with X-ray crystallography and multi-angle light scattering (MALS), to study the oligomer dynamics of GAC in relation to its activation in vitro. To compliment in vitro biophysical studies of GAC activation and inhibition, we employed stable 13C-isotope tracing assays in a model Dbl-inducible cell system to monitor 13C incorporation in TCA cycle intermediates derived from 13C-glutamine. Using dynamic FRET assays, as well as novel GAC mutants that are trapped as constitutive dimer or monomer species, we show that the dimer-to-tetramer transition is indeed directly linked to activation of the enzyme. Subsequently we go on to show that the two previously described allosteric GAC inhibitors, 968 and BPTES, elicit their inhibitory effects through distinctly different mechanisms. Both, however, target specific oligomeric states of GAC: compound 968 preferentially binds to the monomeric form of GAC to disrupt critical oligomer contacts, in stark contrast to BPTES, which binds to and induces tetramer formation by interacting with a loop located near the glutamine binding site. The binding of compound 968 to the monomeric form of GAC was found to induce a conformational change that renders the enzyme inactive, however still able to participate in oligomer formation and dissociation. The consequence of this 968-bound GAC species in vivo was investigated by first showing that 968 treatment of a Dbl-inducible MEF cell line with 968 was capable of reversing Dbl-induced transformation as evidenced by the inhibition of focus formation and proliferation. In this same model cell system, we show that induction of the Dbl-oncogene leads to an increase in glutaminolysis and glutamine fueled anaplerosis by monitoring 13C-enrichment in TCA cycle intermediates derived from 13C-glutamine, and that this increased glutaminolysis is potently inhibited by 968 treatment. These results highlight advances in understanding the mechanism of glutaminase regulation as well as the potential future of small molecules targeting glutamine metabolism as therapeutics. (1) Wang, J.B. et al., (2010) Cancer Cell 18, 207-219 (2) Le, A. et al., (2012) Cell Metabolism 15, 110-121 This project was supported by the Chemical Biology Training Grant (Award Number T32GM008500) from the National Institute of General Medical Sciences and the NIH Citation Format: Clint Stalnecker, Jon Erickson, Sekar Ramachandran, Ralph DeBerardinis, Rick Cerione. Mitochondrial glutaminase regulation and small molecule inhibition of glutamine metabolism. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C42.

Compressive force generation by a bundle of living biofilaments

To study the compressional forces exerted by a bundle of living stiff filaments pressing on a surface, akin to the case of an actin bundle in filopodia structures, we have performed particulate molecular dynamics simulations of a grafted bundle of parallel living (self-assembling) filaments, in chemical equilibrium with a solution of their constitutive monomers. Equilibrium is established as these filaments, grafted at one end to a wall of the simulation box, grow at their chemically active free end, and encounter the opposite confining wall of the simulation box. Further growth of filaments requires bending and thus energy, which automatically limit the populations of longer filaments. The resulting filament sizes distribution and the force exerted by the bundle on the obstacle are analyzed for different grafting densities and different sub- or supercritical conditions, these properties being compared with the predictions of the corresponding ideal confined bundle model. In this analysis, non-ideal effects due to interactions between filaments and confinement effects are singled out. For all state points considered at the same temperature and at the same gap width between the two surfaces, the force per filament exerted on the opposite wall appears to be a function of a rescaled free monomer density ρ1(eff). This quantity can be estimated directly from the characteristic length of the exponential filament size distribution P observed in the size domain where these grafted filaments are not in direct contact with the wall. We also analyze the dynamics of the filament contour length fluctuations in terms of effective polymerization (U) and depolymerization (W) rates, where again it is possible to disentangle non-ideal and confinement effects.

Sequencing of Oligourea Foldamers by Tandem Mass Spectrometry

This study is focused on sequence analysis of peptidomimetic helical oligoureas by means of tandem mass spectrometry, to build a basis for de novo sequencing for future high-throughput combinatorial library screening of oligourea foldamers. After the evaluation of MS/MS spectra obtained for model compounds with either MALDI or ESI sources, we found that the MALDI-TOF-TOF instrument gave more satisfactory results. MS/MS spectra of oligoureas generated by decay of singly charged precursor ions show major ion series corresponding to fragmentation across both CO-NH and N'H-CO urea bonds. Oligourea backbones fragment to produce a pattern of a, x, b, and y type fragment ions. De novo decoding of spectral information is facilitated by the occurrence of low mass reporter ions, representative of constitutive monomers, in an analogous manner to the use of immonium ions for peptide sequencing.

Separation and fractionation of exopolysaccharides from Porphyridium cruentum

In this work the extraction of EPSs from culture media of Porphyridium cruentum, by dialysis, solvent-precipitation with 3 polar alcohols (methanol, ethanol and isopropanol) and membrane separation techniques has been studied. Diafiltration (DF) using a membrane with a 300 kDa molecular weight cut off was the most efficient technique compared to solvent-extraction and dialysis methods. After extraction, EPS fraction was characterized in terms of rheological properties and biochemical content. The product exhibited shear thinning behavior and a critical overlap concentration equal to 0.6 g/L. The monosaccharide composition was investigated after acidic hydrolysis. Xylose, galactose, glucose and glucuronic acid were identified as the main constitutive monomers.

Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.

Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach

Cutins of plant shoots and suberins, mostly present in roots could contribute to significant portions of stable soil organic matter. Their biomarker potential, residing in their unique compositions in different plant types, has been used previously to infer sources of organic matter in sediments. These aliphatic plant biopolyesters contain specific biomarkers, which may be used for tracing their fate in soils and sediments, when combined with stable 13C isotope labelling. In order to evaluate the potential use of cutin and suberin biomarkers as indicators of shoot and root contributions from C3 and C4 plant origins, the objectives of this study were to 1) identify their constitutive monomers, which are specific for shoots and roots of maize (C4) and wheat (C3); 2) evaluate the 13C differences between maize and wheat biomarkers. Mid-chain hydroxy carboxylic acids were mainly found in the aboveground biomass, while α,ω-alkanedioic acids were only present in the roots. The differences in the isotopic composition of the specific monomers between wheat and maize plants (17–18‰ for shoot markers, 19‰ for root markers) were larger than those observed for bulk plant tissues and close to those observed for lignin-derived phenols in other studies. These differences should make it possible to differentiate and quantify the different types and sources of organic matter in sediments and soils. In particular, the molecular and isotopic signatures of cutins and suberins can be used to evaluate the specific dynamics of root vs shoot tissues in soils using C3/C4 chronosequences.

Dimerization is Essential for the Large Step Size of Myosin VI

Myosin VI is an unconventional actin-based motor protein that challenges the classical lever arm hypothesis with its minus-end directed processive movement and unusually large step size, in spite of having a short lever arm with a single IQ domain. The presence of a unique insert (Insert 2) and rearrangement in the converter subdomain can account for its reverse directionality and large powerstroke. However, these adaptations are not sufficient in explaining the 30-36nm step sizes of myosin VI which is typically characteristic of motor proteins with longer lever arms, viz. myosin V with 6 IQ domains. Though it is possible that myosin VI can function either as a monomer or a dimer in cells, based on our studies on the functional properties of the protein, it is likely that a dimeric protein will be a more efficient actin anchor and a processive transporter. We have shown that dimerization of full-length myosin VI can be triggered by cargo binding and the cargo-bound motors walk processively on actin filaments with the expected step size. Our recent studies demonstrate that the region immediately distal to the lever arm of the myosin VI motor (the proximal tail) exists as a three-helix bundle and unfolds upon dimerization of two myosin VI monomers, therefore serving as a lever arm extension in myosin VI. Additionally, our studies show that the medial tail domain is necessary for dimerization since specific mutations in this region create constitutive monomers which are non-processive with no gating properties, indicating that dimerization is necessary for the proper functioning of myosin VI.

Essential Features of a Non-processive Class V Myosin from Budding Yeast for ASH1 mRNA Transport

A feature of most class V myosins is their ability to move processively on actin. The budding yeast Saccharomyces cerevisiae has a non-processive class V myosin, Myo4p, which is a single-headed but high duty cycle motor. Its cellular role is to asymmetrically transport more than 20 different mRNAs, a widely used strategy to polarize a protein within the cell. The most studied mRNA is ASH1, which is moved by Myo4p to the bud tip to repress mating type switching in the daughter cell. Here we determine the features of Myo4p that are necessary for correct localization of ASH1 mRNA to the daughter cell. This process requires the adapter protein She3p, and the mRNA binding protein She2p, which binds ASH1 at specific localization elements called zip codes. Based on a series of chimeric constructs, we showed that the rod region of Myo4p, but not the globular tail, is essential for correct localization of ASH1 mRNA. The rod thus contains the primary binding site for She3p, consistent with our earlier in vitro studies (Hodges et al., 2008). To test if mRNA localization is more efficient when two motors are coupled together, we compared transport by a constitutive dimer of Myo4p/She3p with a constitutive monomer. Correct ASH1 mRNA localization was achieved equally well with both constructs. This may reflect the fact that many mRNAs and thus many motors are part of the translocation complex. Our results show that the most important feature for correct localization is the retention of coupling between all the members of the complex (Myo4p- She3p-She2p-ASH1 mRNA), which is aided by She3p being a tightly bound subunit of Myo4p.

The influence of triethylene glycol derived from dental composite resins on the regulation of Streptococcus mutans gene expression

Bacterial microleakage along the tooth/composite resin dental restoration interface contributes to post-operative sensitivity, recurrent caries, pulp inflammation and necrosis. Studies have confirmed that saliva can catalyze the degradation of constitutive monomers in dental restorative composites, forming biodegradation by-products (BBPs) such as methacrylic acid (MA), and triethylene glycol (TEG). TEG accelerates the growth of Streptococcus mutans, a major etiological agent of dental caries. Restriction fragment differential display polymerase chain reaction (RFDD-PCR) in conjunction with single strand conformation polymorphism (SSCP) was used to identify S. mutans genes with differential expression when grown in the presence of TEG at pH levels 5.5 and 7.0. Quantitative real-time PCR (q-RT PCR) was utilized to study specific gene expression patterns. TEG modulated the expression levels of glucosyltransferase B ( gtfB) (involved in biofilm formation) and yfiV (a putative transcription regulator) in S. mutans. The expression patterns were dependent on the bacterial growth mode (planktonic vs. biofilm) as well as pH (5.5 vs. 7.0). The findings describe the effect of composite resin-derived BBPs on important physiological functions of S. mutans (at BBP concentration levels found in vivo), and indicate the potential influence of BBPs in biofilm formation and microbial survival on surfaces in the oral cavity.