pH‐Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases

A general approach is reported for the design of small‐molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self‐inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH‐responsive glycomimetics targeting human glucocerebrosidase or α‐galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed.     

Cyclooxygenase inhibitors derived from thalidomide

Several N-3,5-dimethylphenylphthalimide analogs possessing more potent cyclooxygenase-inhibiting activity than that of aspirin were prepared during structural development studies based on thalidomide. Substituent effects on the activity were investigated.     

Properties of the bimodal fluorescent protein produced by Photobacterium phosphoreum

A fluorescent protein isolated from the deep-sea luminous bacterium Photobacterium phosphoreum strain bmFP has been purified, cloned and sequenced. The protein is 96.5% identical in amino acid sequence to FP390, the weakly fluorescent flavoprotein encoded by the luxF gene characteristic of Photobacterium species. Similar to FP390, bmFP is a dimer of two homologous subunits binding four FMN-myristate chromophores but has the distinctive feature of emitting a bimodal fluorescence with maxima at about 488 and 517 nm, hence the name bmFP. For both bands of this fluorescence, the excitation spectrum exhibits a peak at 336 nm, not corresponding to its flavin-like absorption spectrum. Heating of bmFP in urea resulted in a decrease in the intensity of the 488 nm band along with the appearance of a new fluorescence peaking at 423 nm, partially reversible upon the removal of the urea. Upon complete denaturation, either by heat or guanidium chloride at 65 degrees C, fluorescence characteristic of both free flavin and this 423 nm species appears. It is speculated that chromophores in different states of protonation, associated with a single protein, are responsible for the unusual spectral properties of bmFP.     

An enzyme immunosensor for IgG

An enzyme immunosensor has been developed for assaying human immunoglobulin G (IgG). The sensor is composed of an oxygen sensoring system and an antibody-binding membrane. The assay procedure involves the competitive immunochemical reaction of the membrane-bound antibody with nonlabeled and catalase-labeled IgG and the electrochemical determination of membrane-bound catalase activity. The analytical result is directly displayed by the output current of the sensor. The sensor exhibited an excellent performance in monitoring specifically human IgG.     

Atomic scattering factors for heavy atoms as investigated by small-angle gas electron diffraction

The experimental examination of the elastic and inelastic scattering factors for tin, iodine and mercury has been performed through the total-intensity     

N-phenylphthalimide-type cyclooxygenase (COX) inhibitors derived from thalidomide: substituent effects on subtype selectivity

Several N-substituted phenylphthalimide and phenylhomophthalimide derivatives with cyclooxygenase (COX)-inhibitory activity were prepared during structural development studies based on thalidomide as a lead compound. Substituent effects on the subtype selectivity were investigated.     

Characterization of catechol‐containing natural thermosetting polymer “urushiol” thin film

Urushi (oriental lacquer) is made from the sap of Rhus vernicifera and consists mainly of the catechol derivative urushiol. Thermal curing of urushiol, unlike other catechol derivatives, is possible because of the unsaturated double bonds in the long hydrocarbon side chain. We described here a simple, efficient method to produce a thermally cross‐linked urushi thin film using iron(II) acetate as the additive. The cured thin films showed robust, flexible feature as confirmed by strain‐induced elastic buckling instability for mechanical measurements (SIEBIMM) and bulging experiments. In contrast, a thin film of poly(dopamine) that is a typical catechol derivative without long hydrocarbon side chain showed brittleness. The long hydrocarbon side chain of urushiol plays an important role for both thermal processability and superior mechanical properties of the material. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3688–3692     

In situ Raman imaging of osteoblastic mineralization

Hydroxyapatite (HA) is synthesized at early stages of bone formation by osteoblasts. Nondestructive observation of early stages of osteoblastic mineralization provides crucial information for biological mechanism of bone formation. Raman microscopy serves as an ideal tool to observe the osteoblastic mineralization process because it shows the chemical information of the sample at a minimally invasive level. In addition, HA is a marker for osteoblastic mineralization, and HA Raman signal is strong enough to identify mineralized spots in osteoblasts. In this research, we visualized the distribution of HA in cultured mouse osteoblasts by Raman imaging and observed the location of the mineralized spots in the culture. We monitored HA Raman signal from osteoblast culture for 3 days after administrating the osteogenic differentiation medium and observed Raman signal associated with HA. We identified mineralized spots of KUSA‐A1 by Raman imaging constructed from the distribution of HA Raman signal. We successfully visualized the distribution of the mineralized spots in the culture of KUSA‐A1. We compared our Raman images with Alizarin red S staining assay, which was a conventional method to evaluate the mineralization process. Raman imaging of the KUSA‐A1 culture visualized the mineralized spots more accurately than Alizarin red S staining assay. Raman imaging of HA serves as a powerful tool to identify the mineralized spots in an in vitro culture of osteogenic lineage cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Metal-binding ability of human prion protein fragment peptides analyzed by column switch HPLC

The structural conversion of the prion protein (PrP) from the normal cellular isoform (PrP(C)) to the posttranslationally modified form (PrP(Sc)) is thought to relate to Cu2? binding to histidine (H) residues. Traditionally, the binding of metals to PrP has been investigated by monitoring the conformational conversion using circular dichroism (CD). In this study, the metal-binding ability of 21 synthetic peptides representing regions of human PrP(C) was investigated by column switch high-performance liquid chromatography (CS-HPLC). The CS-HPLC system is composed of a metal chelate affinity column and an octadecylsilica (ODS) reversed-phase column that together enable the identification of metal-binding regardless of conformational conversion. Synthetic peptides were designed with respect to the position of H residues as well as the secondary structure of human PrP (hPrP). The ability of the octapeptide (PHGGGWGQ)-repeating region (OP-repeat) to bind metals was analyzed by CS-HPLC and supported by CD analysis, and indicated that CS-HPLC is a reliable and useful method for measuring peptide metal-binding. Peptides from the middle region of hPrP showed a high affinity for Cu2?, but binding to Zn2?, Ni2?, and Co2? was dependent on peptide length. C-Terminal peptides had a lower affinity for Cu2?, Zn2?, Ni2?, and Co2? than OP-repeat region peptides. Interestingly, hPrP193-230, which contained no H residues, also bound to Cu2?, Zn2?, Ni2?, and Co2?, indicating that this region is a novel metal-binding site in the C-terminal region of PrP(C). The CS-HPLC method described in this study is useful and convenient for assessing metal-binding affinity and characterizing metal-binding peptides or proteins.