Dynamics and organization of MAP kinase signal pathways

In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MAPK activation cascade mediating this signal is made up of Ste11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK-related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades. Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway. Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7-P368 has higher basal activity than the wild-type enzyme but still requires Ste11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7-P368 is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7-P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission.     

Methylome Profiling in Fabry Disease in Clinical Practice: A Proof of Concept

Anderson–Fabry disease (FD) is an X-linked disease caused by a functional deficit of the α-galactosidase A enzyme. FD diagnosis relies on the clinical manifestations and research of GLA gene mutations. However, because of the lack of a clear genotype/phenotype correlation, FD diagnosis can be challenging. Recently, several studies have highlighted the importance of investigating DNA methylation patterns for confirming the correct diagnosis of different rare Mendelian diseases, but to date, no such studies have been reported for FD. Thus, in the present investigation, we analyzed for the first time the genome-wide methylation profile of a well-characterized cohort of patients with Fabry disease. We profiled the methylation status of about 850,000 CpG sites in 5 FD patients, all carrying the same mutation in the GLA gene (exon 6 c.901C>G) and presenting comparable low levels of α-Gal A activity. We found that, although the whole methylome profile did not discriminate the FD group from the unaffected one, several genes were significantly differentially methylated in Fabry patients. Thus, we provide here a proof of concept, to be tested in patients with different mutations and in a larger cohort, that the methylation state of specific genes can potentially identify Fabry patients and possibly predict organ involvement and disease evolution.     

Polymer swelling 2: A restudy of poly(styrene-co-divinylbenzene) swelling in terms of the cross-link density1

A very convenient and reliable gravimetric method was developed for measuring swelling of poly(styrene-co-divinylbenzene) in particulate form. The method is based on the gravimetric procedure reported earlier² for monitoring liquid uptake by thin (<0.3 mm) microporous composite films, consisting of swellable particulate (80% by weight) enmeshed in poly(tetrafluoroethylene) microfibers (20%). The swellability S (in milliliter of liquid absorbed per gram of polymer in equilibrium with excess liquid) for six sty-co-DVB polymers with crosslink densities ranging from 0.01 to 0.12 was measured in 19 organic liquids. In each study of S as a function of the relationship was given by where is the average number of carbon atoms in the “backbone” of the polystyrene segments between cross-link junction, C is the relative swelling power of the liquid, and is the critical cross-link density above which S is equal to zero.     

Picosecond GaAs photoconductors on silicon substrates for localintegration with silicon devices and circuits

A fabrication procedure for local integration of GaAs photoconductive devices with processed silicon circuits is discussed. The process allows isolated regions of GaAs to be epitaxially grown by MBE at temperatures which are compatible with already processed silicon circuits with first-level metallization. GaAs photoconductors with 15-μm gap lengths fabricated on silicon substrates have exhibited >16-mA sampling-oscilloscope-limited responses, with electrical pulse widths less than 20 ps as determined by autocorrelation measurements     

Guar gum methyl ethers. Part I. Synthesis and macromolecular characterization

Guar gum (GG) has been partially methyl-etherified under heterogeneous reaction conditions. The resulting products, (methyl ether guar: MG) with different degrees of substitution, have been characterized by means of viscosity, ¹H NMR, and FTIR measurements. The introduction of methoxyl groups along the polysaccharidic chains reduces the hydrogen bonding sites on the guar backbone reducing primarely the extent of hydrogen bonding between guar macromolecules, hence their aggregation tendency. A comparative analysis of Mark–Houwink–Sakurada parameters and of the characteristic ratio (C_∞) of GG and MG samples in aqueous solution has been carried out using the Burchard–Stockmayer–Fixman method for flexible and semiflexible chains. The MG chains exhibit more flexibility than those of native guar gum which is traceable to a disruption of intrachain hydrogen bonds.     

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/w_PLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 μm and its internal diameter was 2.61 ± 0.05 mm. The graft’s antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.     

Polymer drying. X. A recondsideration of the kinetics of evaporation from polymer–liquid systems during the interval of transition from the rubbery to a glassy state

This kinetic restudy of the physical changes that occur during evaporation-induced transition from the rubbery to a glassy state of polystyrene–liquid systems shows that such transitions occur via two mechanistic pathways. The first is random nucleation of microdomains of self-associated polymer segments owing to a time-dependent logarithmic decrease in the number of adsorbed volatile molecules per phenyl group of residual mobile polymer segments. The second is a therodynamically driven self-association of adjacent monomer units with concomitant explusion of the adsorbed molecules, which appears to propagate via a “dominolike” chain reaction. Conceptually this is a three-dimensional “zippering-up” of suitably close polymer segments to produce the corresponding macrostructural network of self-associated polymer. The kinetics of the latter is zero-order, and this dominates the overall kinetics of evaporation during the latter portion of the transition interval, presumably owing to changes in entropy of the system as it progresses from the mobility characteristic of the rubbery state to the rigidity characteristic of a glassy state. © 1994 John Wiley & Sons, Inc.     

An anion-permeable polymeric coating for the protection of a semiconductor photoelectrode against decomposition

An anion-permeable polymeric film has been applied to the surfaces of cadmium sulfide photoanodes to decrease the rate of electrode corrosion which is observed during the photoelectro-chemical oxidation of aqueous species. Although the rate of surface corrosion was reduced, film adhesion problems were observed.