Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK _a of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease. Zhen-Dan Shi and Omid Motabar contributed equally to this work     

Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency

Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes, and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. To fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved (11)CXX(14)C active site were chemically synthesized. Grx3, Grx3(C11U), and Grx3(C14U) exhibited redox potentials of -194, -260, and -275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (-309 mV) and thioredoxin (Trx) (-270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a "central atom" in the exchange reaction. The 10(2)-10(4)-fold increase in the rate of Trx reduction by the seleno-Grx3 analogues demonstrates that oxidoreductases containing either selenenyl-sulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts.     

Thermal and chemical stability of diphenylalanine peptide nanotubes: implications for nanotechnological applications

The diphenylalanine peptide, the core recognition motif of the beta-amyloid polypeptide, efficiently self-assembles into discrete, well-ordered nanotubes. Here, we describe the notable thermal and chemical stability of these tubular structures both in aqueous solution and under dry conditions. Scanning and transmission electron microscopy (SEM and TEM) as well as atomic force microscopy (AFM) revealed the stability of the nanotubes in aqueous solution at temperatures above the boiling point of water upon autoclave treatment. The nanotubes preserved their secondary structure at temperatures up to 90 degrees C, as shown by circular dichroism (CD) spectra. Cold field emission gun (CFEG) high-resolution scanning electron microscope (HRSEM) and thermogravimetric analysis (TGA) of the peptide nanotubes after dry heat revealed durability at higher temperature. It was shown that the thermal stability of diphenylalanine peptide nanotubes is significantly higher than that of a nonassembling dipeptide, dialanine. In addition to thermal stability, the peptide nanotubes were chemically stable in organic solvents such as ethanol, methanol, 2-propanol, acetone, and acetonitrile, as shown by SEM analysis. Moreover, the acetone environment enabled AFM imaging of the nanotubes in solution. The significant thermal and chemical stability of the peptide nanotubes demonstrated here points toward their possible use in conventional microelectronic and microelectromechanics processes and fabrication into functional nanotechnological devices.     

Multiphase thermoplastic hybrid for controlled release of antimicrobial essential oils in active packaging film

Active packaging, a new technology concept in the field of food packaging, has been introduced in recent years in order to provide quality and safety, as well as extend the shelf life of food products. Antimicrobial (AM) agents can be incorporated directly into the active packaging and migrate in a controlled manner to the headspace between the food and the package, inhibiting bacteria growth on the food surface. Naturally derived AM agent, such as essential oils (EOs), has received considerable attention for food preservation purposes, because of their effective AM activity against various bacteria and fungi. In the present study, AM active film systems based on polypropylene/polyamide blends, montmorillonite nanoclays, and thymol EO were produced to investigate the feasibility of controlling the release rate of thymol from food packaging systems. Selective localization of thymol in a specific phase in the system that derives from thermodynamic affinity was assumed to be useful in controlling its migration rate from the film to the headspace. EO retention in the film under two different time conditions was measured by spectroscopic analysis. The release rate of EO was determined using Gas chromatography technique and analyzed by diffusion model approach. Inhibition of bacterial growth was periodically tested for Listeria and Escherichia coli bacteria. This study confirms the thermodynamic affinity of polyamide phase with thymol that has a positive effect in retaining the EO. Results show controlled AM behavior of the active packaging films, based on various blend compositions. Copyright © 2016 John Wiley & Sons, Ltd.     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

Central Value Of Automorphic <Emphasis Type="Italic">L</Emphasis>-Functions

We prove a generalization to the totally real field case of the Waldspurger’s formula relating the Fourier coefficient of a half integral weight form and the central value of the L-function of an integral weight form. Our proof is based on a new interpretation of Waldspurger’s formula as a combination of two ingredients – an equality between global distributions, and a dichotomy result for theta correspondence. As applications we generalize the Kohnen–Zagier formula for holomorphic forms and prove the equivalence of the Ramanujan conjecture for half integral weight forms and a case of the Lindel?f hypothesis for integral weight forms. We also study the Kohnen space in the adelic setting. The first author was partially supported by NSF grant DMS-0070762. The second author was partially supported by NSF grant DMS-0355285. Received: July 2005 Accepted: August 2005     

The Mordell-Lang conjecture for function fields

We give a proof of the geometric Mordell-Lang conjecture, in any characteristic. Our method involves a model-theoretic analysis of the kernel of Manin's homomorphism and of a certain analog in characteristic .

     

Structure-Activity and Mechanism Studies on Silicon Phthalocyanines with Plasmodium falciparum in the Dark and Under Red Light

Syntheses for the new photosensitizers HOSiPc-OSi(CH₃)₂(CH₂)₃N(CH₂)₁ or ₃(CH₃)₂, Pc 34 and Pc 25, have been developed and the order of activity of these photosensitizers and the previously reported photosensi-tizer Pc 4, HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂, in the dark and with broad-band red light toward Plasmodium falciparum in red blood cell (RBC) suspensions has been studied. The order of activity has been found to be Pc 4 Pc 34 Pc 25. Thus, the activity of the photosensitizers under both sets of conditions is inversely proportional to the length of their terminal amino alkyl chains. The 50% inhibition dye concentration (IC₅₀) in the dark for the parasites in RBC suspension with Pc 4 is 24 nM and the dye concentration and light fluence that yield:3 log₁₀ of parasite inactivation with Pc 4 are 2 mM and 3 J/cm², respectively. The synthesis of DNA and proteins by the parasites in culture was strongly inhibited by Pc 4 in the dark while parasite lactate dehydrogenase (pLDH) activity was unaffected. With Pc 4 and light, DNA and protein synthesis of the parasites in culture was strongly inhibited, pLDH activity of the parasites was moderately inhibited and ribosome density of the parasite cells was reduced. Gel electrophoresis studies showed that synthesis of all parasite proteins was inhibited to a similar extent. These results suggest that Pc 4 both in the dark and with light inactivates the cells by disturbing their machinery for the synthesis of not just one but a whole series of proteins. It is concluded that Pc 4 and light may be able to serve as a practical sterilization combination not only for HIV and other viruses but also for malaria parasites in RBC concentrates, and that Pc 4 by itself may have potential as a chemotherapeutic agent toward malaria.     

Bounded variation of {V n } and its limit

This note studies relations between limV _n and the lower long-run average value ( \(\underline V \) ) in dynamic programming. It is shown that a certain bounded variation conditions of {V _n } implies that limV _n = \(\underline V \) .