Conformational dynamics of amyloid-β (16–22) peptide in aqueous ionic liquids

Molecular dynamics simulations of amyloid-β (16–22) peptide dimer in water as well as at two different experimentally studied concentrations of hydrated ionic liquids (ILs), ethylammonium mesylate (EAM), ethylammonium nitrate (EAN), and triethylammonium mesylate (TEAM), were carried out employing an umbrella sampling method. We used the average Ψ angle of the peptide backbone as the reaction coordinate to observe the conformational changes of a peptide dimer. Secondary structural element values were calculated for the peptide dimer along the reaction coordinate to see the transition of the peptide dimer between β-sheet and α-helix conformations. We observe the β-sheet conformation as the global minimum on the free energy surfaces in both EAM and EAN ILs at both the concentrations and at a low concentration of TEAM. However, we observe α-helix conformation as the global minimum at a high concentration of TEAM. Our results are in good correlation with the experimental findings. We calculated the average number of intramolecular and intermolecular hydrogen bonds of α-helix and β-sheet conformations in all solutions, and they are in correlation with the secondary structure element values. To understand the peptide–IL interactions, atom–atom radial distribution functions of cation, anion, and water around amide oxygen and hydrogen atoms were calculated. The solvent-accessible surface area of the peptide dimer was calculated to understand the exposure of the peptide towards the solvent during conformational changes. Finally, van der Waals (vdW) and Coulomb interaction energies were calculated between peptide–cation, peptide–anion, and peptide–water to understand the stability of conformations in different concentrations. We find that the TEA cation has more vdW interaction energy compared to Coulomb interaction energy with peptide in 70% (w/w) TEAM, which mimics a membrane-like environment to induce α-helix conformation rather than β-sheet conformation.

Molecular dynamics simulations of amyloid-β (16–22) peptide dimer at two different experimentally studied concentrations of hydrated ethylammonium mesylate, ethylammonium nitrate, and triethylammonium mesylate were carried out employing an umbrella sampling method.     

Replica exchange molecular dynamics simulation of the coordination of Pt(ii)-Phenanthroline to amyloid-β

We report replica exchange molecular dynamics (REMD) simulations of the complex formed between amyloid-β peptides and platinum bound to a phenanthroline ligand, Pt(phen). After construction of an AMBER-style forcefield for the Pt complex, REMD simulation employing temperatures between 270 and 615 K was used to provide thorough sampling of the conformational freedom available to the peptide. We find that the full length peptide Aβ42, in particular, frequently adopts a compact conformation with a large proportion of α- and 3,10-helix content, with smaller amounts of β-strand in the C-terminal region of the peptide. Helical structures are more prevalent than in the metal-free peptide, while turn and strand conformations are markedly less common. Non-covalent interactions, including salt-bridges, hydrogen bonds, and π-stacking between aromatic residues and the phenanthroline ligand, are common, and markedly different from those seen in the amyloid-β peptides alone.

Replica exchange molecular dynamics are used to explore the conformational freedom of amyloid-βbound to Pt(phenanthroline), highlighting important differences in secondary and tertiary structure from the metal-free peptide.     

Oxygen Equilibrium Characteristics of Abnormal Hemoglobins: Hirose (α2β237Ser), L Ferrara (α247Glyβ2), Broussais (α290Asnβ2), and Dhofar (α2β258Arg)

The oxygen equilibrium characteristics of four structural variants of hemoglobin A were correlated with their amino acid substitutions.Hemoglobin Dhofar, in which the proline at E2(58)beta is replaced by arginine, had normal oxygen equilibrium characteristics.Hemoglobin L Ferrara. in which the aspartic acid at CD5(47)alpha is replaced by glycine, and hemoglobin Broussais, in which the lysine at FG2(90)alpha is replaced by asparagine, both showed a slightly elevated oxygen affinity; nevertheless both demonstrated a normal heme-heme interaction and a normal Bohr effect.Hemoglobin Hirose, in which the tryptophan at C3 (37)beta is replaced by serine, showed abnormalities of all oxygen equilibrium characteristics; i.e., increased oxygen affinity, diminished heme-heme interaction, and reduced Bohr effect.These results suggest that aspartic acid at CD5(47)alpha and lysine at FG2(90)alpha are involved in the function of the hemoglobin molecule, despite the fact that these positions are not located directly in the heme or the alpha-beta-contact regions.Tryptophan at C3(37)beta is located at contact between alpha(1)- and beta(2)-subunits. It is suggested that the substitution by serine might disturb the quarternary structure of the mutant hemoglobin molecule during transition from oxy-form to deoxy-form resulting in an alteration of the heme function.     

The polysaccharides from Grifola frondosa attenuate CCl4-induced hepatic fibrosis in rats via the TGF-β/Smad signaling pathway

The TGF-β1/Smad signaling pathway has been linked to hepatic fibrosis. Previous studies have shown that yellow polysaccharide can prevent the development of hepatic fibrosis. However, it is unclear whether the polysaccharide affects the TGF-β1/Smad signaling pathway. In this experiment, 50 experimental rats were randomly divided into a normal control group, model group, low GFP dose group (50 mg kg⁻¹), medium GFP dose group (100 mg kg⁻¹), and high GFP dose group (200 mg kg⁻¹). A cirrhotic portal hypertension rat model was established by a CCl₄ compound method. After 12 weeks of intragastric administration, the liver index of the medium dose and high dose group was significantly lower than that of the model group. The hepatic fibrosis lesions of rats in each dose group were improved to different extents, and the effect was most significant in the high dose group. The contents of ALT, AST, TBIL and CIV, PCIII, LN and HA in serum were significantly decreased. The activity of SOD and GSH-Px in the liver tissue of GFP medium and high dose groups was significantly increased and the content of MDA was significantly decreased. The contents of TNF-α, IL-1β and IL-6 were significantly decreased. The western blot results showed that the expressions of p-Smad 2/3, Smad4, PAI-1, Imp7 and Imp8 in medium dose and high dose groups were significantly lower than those in the model group, while the expression of Smad7 was significantly higher than that of the model group. The GFP-treated group was able to reduce the expression level of mi R-154 in liver tissue and increase the expression level of miR-146a. GFP has a significant intervention effect on rat hepatic fibrosis, and its mechanism may inhibit the progression of hepatic fibrosis by inhibiting oxidative stress and inflammatory response and regulating TGF-β1/Smad signaling pathway and mi RNA expression.

The TGF-β1/Smad signaling pathway has been linked to hepatic fibrosis.     

Estrogen Protects Lenses against Cataract Induced by Transforming Growth Factor-β (TGFβ)

Cataract, already a major cause of visual impairment and blindness, is likely to become an increasing problem as the world population ages. In a previous study, we showed that transforming growth factor-β (TGFβ) induces rat lenses in culture to develop opacities and other changes that have many features of human subcapsular cataracts. Here we show that estrogen protects against cataract. Lenses from female rats are more resistant to TGFβ-induced cataract than those from males. Furthermore, lenses from ovariectomized females show increased sensitivity to the damaging effects of TGFβ and estrogen replacement in vivo, or exposure to estrogen in vitro, restores resistance. Sex-dependent and estrogen-related differences in susceptibility to cataract formation, consistent with a protective role for estrogen, have been noted in some epidemiological studies. The present study in the rat indicates that estrogen provides protection against cataract by countering the damaging effects of TGFβ. It also adds to an increasing body of evidence that hormone replacement therapy protects postmenopausal women against various diseases.     

USP25 inhibition ameliorates Alzheimer’s pathology through the regulation of APP processing and Aβ generation

Down syndrome (DS), or trisomy 21, is one of the critical risk factors for early-onset Alzheimer’s disease (AD), implicating key roles for chromosome 21–encoded genes in the pathogenesis of AD. We previously identified a role for the deubiquitinase USP25, encoded on chromosome 21, in regulating microglial homeostasis in the AD brain; however, whether USP25 affects amyloid pathology remains unknown. Here, by crossing 5×FAD AD and Dp16 DS mice, we observed that trisomy 21 exacerbated amyloid pathology in the 5×FAD brain. Moreover, bacterial artificial chromosome (BAC) transgene–mediated USP25 overexpression increased amyloid deposition in the 5×FAD mouse brain, whereas genetic deletion of Usp25 reduced amyloid deposition. Furthermore, our results demonstrate that USP25 promoted β cleavage of APP and Aβ generation by reducing the ubiquitination and lysosomal degradation of both APP and BACE1. Importantly, pharmacological inhibition of USP25 ameliorated amyloid pathology in the 5×FAD mouse brain. In summary, we identified the DS-related gene USP25 as a critical regulator of AD pathology, and our data suggest that USP25 serves as a potential pharmacological target for AD drug development.     

LiCl-promoted amination of β-methoxy amides (γ-lactones)

An efficient and mild method has been developed for the amination of β-methoxy amides (γ-lactones) including natural products michelolide, costunolide and parthenolide derivatives by using lithium chloride in good yields. This reaction is applicable to a wide range of substrates with good functional group tolerance. Mechanism studies show that the reactions undergo a LiCl promoted MeOH elimination from the substrates to form the corresponding α,β-unsaturated intermediates followed by the Michael addition of amines.

The amination of β-methoxy amides (γ-lactones) including natural products michelolide, costunolide and parthenolide derivatives were first developed by using lithium chloride.

The formation of carbon–nitrogen bonds remains one of the most fundamental and widely practiced reactions in organic synthesis, due to the prevalence of this functionality in the preparations of functional molecules in pharmaceutical chemistry, biochemistry and material sciences.¹ Various synthetic methodologies have been developed to form C(sp²)-N bonds, including the Goldberg reaction,² Buchwald–Hartwig reaction,³ imine reduction⁴ and the nucleophilic addition of carbon-nucleophiles to imine derivatives.⁵ Meanwhile, the formations of C(sp³)-N bonds can be achieved by reductive amination, which involves the conversion of a carbonyl group to an amine via an imine intermediate, such as Eschweiler–Clarke reaction⁶ and Borch reductive amination.⁷ Nucleophilic substitution of alkyl(pseudo)halides with amines (amine alkylation) serves as one direct strategy for the preparation of alkylamines, while the necessity of pre-installation of the halogen atoms and the production of stoichiometric inorganic salt wastes are considered as two main drawbacks for its application in large scale industrial synthesis.⁸Methoxy as the leaving group in the amination reactions has recently attracted the attention of organic chemists. For instance, Chiba and coworkers reported a method for the nucleophilic amination of methoxy arenes,⁹ which was achieved by using sodium hydride (NaH) in the presence of lithium iodide (LiI) through a concerted nucleophilic aromatic substitution pathway (Fig. 1a).¹⁰ Kondo and coworkers demonstrated that the organic superbase t-Bu-P4 efficiently catalyzes the amination of methoxy(hetero)arenes with the amine nucleophiles (Fig. 1b).¹¹ The t-Bu-P4 is also suitable to catalyze the amination of β-(hetero)arylethyl ethers with amines to synthesize β-(hetero)arylethylamines (Fig. 1c).¹² Sun and coworkers reported that C–S bond cleavage to access N-substituted acrylamide and β-aminopropanamide(Fig. 1d).¹³Open in a separate windowFig. 1Amination reactions of methyl ethers.Recently, we described the application of a CuBr–LiCl composite for the short-chain alkoxylation of aryl bromides.¹⁴ During that course of study, the single-shell lithium ion was found to embrace a unique affinity for oxygen and can be used as an additive to activate C–O bond and facilitate the nucleophilic reaction. On the basis of this study, we herein present the synthesis of β-amino amides (γ-lactones) via the elimination of methoxy group followed by Michael addition of an amine, that was promoted by LiCl in good yields under conventional conditions.We initiated our study with the reaction of 3-methoxy-N-phenylpropanamide 1a and piperidine 2a in the presence of lithium salts (

NH2-MIL-88B (FeαIn1−α) mixed-MOFs designed for enhancing photocatalytic Cr(vi) reduction and tetracycline elimination

Aiming at solving the issue of wastewater purification, this work synthesized NH₂-MIL-88B (Fe_αIn_1−α) photocatalysts by a simple one-pot method, which was employed for photocatalytic reduction of Cr(vi) and oxidation of TC-HCl. Compared with traditional NH₂-MIL-88B (Fe) photocatalysts, NH₂-MIL-88B (Fe_0.6In_0.4) displayed excellent photocatalytic performance; the photocatalytic redox rate for Cr(vi) and TC-HCl reached 86.83% and 72.05%, respectively. The good photocatalytic performance might be attributed to the metal-to-metal charge transition (MMCT) between Fe–O clusters and In–O clusters formed by doping In(iii) into NH₂-MIL-88B (Fe), which provides effective active sites for the photocatalytic reduction and oxidation routes. Besides, the synergistic effect of the ligand-to-metal charge transition (LMCT) and MMCT expands the separation and transfer of photogenerated carriers and inhibits the recombination of electron–hole pairs, thus effectively improving the photocatalytic performance. Therefore, this work could provide a new method for the construction of mixed metal MOFs for the photocatalytic degradation of pollutants.

Aiming at solving the issue of wastewater purification, this work synthesized NH₂-MIL-88B (Fe_αIn_1−α) photocatalysts by a simple one-pot method, which was employed for photocatalytic reduction of Cr(vi) and oxidation of TC-HCl.     

Counterselection against Dμ Is Mediated through Immunoglobulin (Ig)α-Igβ

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (Dμ). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Igα-Igβ, whereas the mechanism for counterselection against Dμ has not been determined. We have examined the role of the Igα-Igβ signal transducers in counterselection against Dμ using mice that lack Igβ. We found that Dμ expression is not selected against in developing B cells in Igβ mutant mice. Thus, the molecular mechanism for counterselection against Dμ in pre-B cells resembles positive selection in that it requires interaction between mDμ and Igα-Igβ.     

THE CELLULAR ORIGIN OF HUMAN IMMUNOGLOBULINS (γ2, γ1M, γ1A)

A study was made of the cellular origin of human immunoglobulins (γ₂, γ_1M, γ_1A). The results indicated that two closely related families of cells form immunoglobulins in human lymphoid tissue: germinal (reticular) centers and plasma cells. Thus their cellular origin in addition to their known antigenic relations further justifies placing the immunoglobulins in one family of proteins. Immunoglobulins were also formed to a small extent in primitive reticular cells which resembled those of germinal centers but were separated from them. Possibly such cells were undergoing transition to the much more numerous plasma cells with which they were commonly associated. The mantles of small lymphocytes which surrounded germinal centers did not contain detectable quantities of immunoglobulins. While in general only one type of immunoglobulin was present in an individual cell or germinal center, γ₂- and γ_1M-globulin were identified on occasion in the same plasma cell and germinal center. A peculiarity of the fetal thymus gland was the presence of immunoglobulin, mainly γ_1M, in a small number of cells of small and intermediate size and primitive reticular appearance and in Hassall's corpuscles.     

Synthesis of 1-(β-coumarinyl)-1-(β-indolyl)trifluoroethanols through regioselective Friedel–Crafts alkylation of indoles with β-(trifluoroacetyl)coumarins catalyzed by Sc(OTf)3

A highly efficient Friedel–Crafts alkylation of indole derivatives with β-(trifluoroacetyl)coumarins using Sc(OTf)₃ as a catalyst has been developed, which gives regioselective 1,2-adducts to afford 1-(β-coumarinyl)-1-(β-indolyl)trifluoroethanols. A series of tertiary trifluoroethanols containing different indole and coumarin groups were synthesized in moderate to excellent yields (up to 95%) in the presence of 5 mol% catalyst in a short time (only 2 minutes at least). A mechanism of the reaction, in which the trace amount of water plays the role of proton transfer in catalyzing circulation was proposed and confirmed.

A Friedel–Crafts alkylation of indoles with β-(trifluoroacetyl)coumarins catalyzed by Sc(OTf)₃ to afford 1-(β-coumarinyl)-1-(β-indolyl)trifluoroethanols in a short time and high yield was developed.     

Blockade of clearance of immune complexes by an anti-F(cγ) receptor monoclonal antibody

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcγR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.     

Development of an MRI contrast agent for both detection and inhibition of the amyloid-β fibrillation process

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was synthesised as an MRI contrast agent for detecting the amyloid-β (Aβ) fibrillation process. Gd-DO3A-Comp.B inhibited Aβ aggregation significantly and detected the fibril growth at 20 μM of Aβ with 10 μM of probe concentration by T₁-weighted MR imaging.

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was developed to significantly inhibit the amyloid-β (Aβ) aggregation and detect the fibril growth by T₁-weighted MR imaging.

A significant increase of Alzheimer''s disease (AD) patients urges the development of therapeutic and diagnostic technology.¹ As with the therapeutic development, diagnostic technology also faces several obstacles. To date, the definite diagnosis of AD relies on the histopathological data of post-mortem.^2,3 The non-invasive imaging technology targeting AD biomarkers such as amyloid β (Aβ) could provide phenotypical diagnostics, although the development of Aβ probes still remains challenging. Several contrast agents for single photon emission computed tomography (SPECT) and positron emission tomography (PET) such as Florbetapir-¹⁸F and Pittsburgh compound-B ([¹¹C]PiB) were developed as efficient tracers in mild cognitive impairment patients.^4,5 However, PET- and SPECT-based diagnostics require injection of radioactive probes, which cannot be measured frequently due to radiation exposure and limited availability of facilities. They also provide limited information on the anatomic profile of biomarkers due to their low spatial resolution and imprecise microscopic localization.⁶ In contrast, magnetic resonance imaging (MRI) contrast agents could quantify the Aβ accumulation in the anatomic brain image.⁷Several reported MRI contrast agents using gadolinium (Gd) complexes demonstrate potential use of Aβ detection. A clinically approved contrast agent, Gd(iii) diethylenetriaminepentaacetic acid (Gd-DTPA) complex accumulates in brain after opening the blood–brain barrier (BBB) by using mannitol and detects Aβ deposits in the mice AD-model.⁸ To improve the selectivity, Gd complexes were conjugated with compounds binding to Aβ such as Pittsburgh compound B (Gd-DO3A-PiB) which also serves as an approach for increasing MRI sensitivity.^9,10 An α,β-unsaturated ketone compound curcumin has been widely reported as an Aβ probe due to its ability to bind the hydrophobic site of Aβ.^11,12 Allen et al. firstly reported the direct conjugation of curcumin with Gd-DTPA which binds to Aβ with four times higher relaxivity than free Gd-DTPA.¹³ Furthermore, a polymalic acid-based nanoparticle covalently linked with curcumin and Gd-DOTA could also detect Aβ in human brain specimen by MRI.¹⁴ These previous studies demonstrate that the curcumin structure has significant potential for the development of MRI contrast agents for AD diagnosis.Previously, we reported a curcumin derivative, compound B, possesses 100-times stronger inhibitory activity of Aβ aggregation than curcumin on the basis of thioflavin T (ThT) competitive binding assay.^15,16 According to this result, we designed curcumin-based Gd probes for the detection and inhibition of Aβ (Fig. 1A–C). We hypothesized that these probes could accelerate proton longitudinal relaxation depending on the fibrillation stage of Aβ, because molecular tumbling rate of the Gd complexes becomes slower (Fig. 1A).¹⁷ As a result, the probes permit the detection of Aβ by longitudinal relaxation time (T₁)-weighted imaging. This mechanism could also be utilized to estimate the inhibitory activity of the probes by T₁-based analysis (Fig. 1B). The curcumin and compound B were directly conjugated with the macrocyclic DO3A ligand through the propylamine linker to obtain Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively (Fig. 1C).Open in a separate windowFig. 1(A) A probe concept that produces T₁ in a dependent manner of Aβ fibrillation process. (B) Inhibitor-based probes that cause moderate T₁ decreases due to inhibitory activity of fibrillation. (C) The chemical structures of the synthesized Gd probes for Aβ detection and inhibition.Gd-DO3A-Cur and Gd-DO3A-Comp.B were synthesized according to Scheme 1 (detail in Scheme S1, ESI^†). The compound 5a and 5b, which have asymmetric curcumin derivatives containing carboxylic acid group, were synthesized by three step reactions. Amide bond formation with DO3A(tBu)₃-propylamine ligand¹⁸ by condensation reaction afforded compound 7a and 7b. The tert-butyl groups were deprotected by trifluoroacetic acid producing compound 8a and 8b. The complexation was performed with GdCl₃·6H₂O by adjusting the reaction pH to 7, giving 43 and 41% yields of Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively. The T₁ relaxivities (r₁) of the curcumin-based Gd probes were estimated by T₁ measurement using a 1 tesla NMR relaxometry (Fig. S1, ESI^†). For the comparison, we synthesized Gd-DO3A-Chal which is a reported probe for Aβ.¹⁹ The r₁ of Gd-DO3A-Comp.B, Gd-DO3A-Cur, and Gd-DO3A-Chal were 7.1, 6.1 and 5.3 mM⁻¹ s⁻¹, respectively. These r₁ values are higher than that of clinically approved Gd-DOTA (3.9 mM⁻¹ s⁻¹).²⁰ The molecular weight of Gd-DO3A-Comp.B and Gd-DO3A-Cur is almost two times larger than that of Gd-DOTA. Because the r₁ increases approximately linearly with molecular weight in low magnetic field,¹⁷ the high r₁ values of Gd-DO3A-Comp.B and Gd-DO3A-Cur might be mainly attributed to their high rotational correlation time, rather than the high number of coordinated water molecules. The r₁ of Gd-DO3A-Chal was comparable to the value reported previously.¹⁹Open in a separate windowScheme 1Synthetic scheme of Gd-DO3A-Cur and Gd-DO3A-Comp.B. (a) B(OH)₃, morpholine, DMF, 100 °C, 10 min. (b) 3a/3b, B(OH)₃, morpholine, DMF, 100 °C, 10 min. (c) TFA, DCM. (d) DO3A(tBu)₃-propylamine ligand, PyBOP, HOBt, Et₃N, DMF. (e) 7a/7b, TFA, DCM. (f) GdCl₃·6H₂O, NaOH, H₂O.We evaluated the inhibitory effect of three probes toward Aβ aggregation by Congo red assay.²¹ After 24 h incubation of 20 μM Aβ with 10 μM probe, Gd-DO3A-Comp.B showed the lowest fluorescence intensity, indicating the strongest inhibitory activity followed by Gd-DO3A-Cur (Fig. 2A). As the comparison, the reported MRI agents, Gd-DO3A-Chal showed slight inhibitory activity. The inhibitory effect was further evaluated by transmission electron microscopy (TEM) with negative staining (Fig. 2B). In the absence of the probes, Aβ formed huge and massive fibril similar to the typical morphology of Aβ fibril.²² The TEM images of Aβ with Gd-DO3A-Comp.B showed the presence of white spheres below 10 nm, demonstrating that Gd-DO3A-Comp.B strongly inhibits Aβ aggregation. In fact, the fibril growth stopped at a stage of oligomer formation. Lower inhibitory activity of Gd-DO3A-Cur was also found to provide a shortened worm-like fibril, which is the typical morphology of Aβ exposed to curcumin.²³ In contrast, the small amount of white spheres and partial fibril disruption were found in the image of Aβ with Gd-DO3A-Chal. In comparison with a reported Gd-DTPA-curcumin possessing inhibitory activity starting at 50 μM, Gd-DO3A-Comp.B possessed stronger inhibition of Aβ aggregation at 10 μM.²⁴ The MTT assay using Neuro 2a cells showed that IC₅₀ of Gd-DO3A-Cur and Gd-DO3A-Comp.B. were more than 500 μM, indicating that these compounds did not possess significant cytotoxicity (Fig. S2, ESI^†).Open in a separate windowFig. 2Inhibitory effect of the Gd probes toward Aβ aggregation measured by Congo red assay (A) and negative staining TEM images (B). The Gd probes were co-incubated with monomeric Aβ for 24 h in PBS at pH 7.4. [Gd] = 10 μM, [Aβ] = 20 μM. Scale bars = 100 nm.To detect fibrillation process by NMR relaxometry, we measured T₁ of the probe mixture with Aβ which were pre-incubated for 1, 3, 6, 12, and 24 h to make it form the fibrils of different growth stages (Fig. 3A and B). The T₁ of Gd-DO3A-Comp.B solution decreased with pre-incubation time of Aβ, demonstrating that the Gd-DO3A-Comp.B can detect Aβ fibril depending on the growth stage (Fig. 3B). Lower T₁ involved with Aβ growth could be caused by the reduction in tumbling rate of the Gd complex.²⁵ We also co-incubated the probes with the Aβ monomer and monitored T₁ changes over the incubation time (Fig. 3A, B and S3, ESI^†). Interestingly, the Gd-DO3A-Comp.B did not cause significant T₁ decreases even after 24 hours co-incubation with Aβ monomers, demonstrating that Gd-DO3A-Comp.B has a strong inhibitory effect on fibril formation and the inhibition can be monitored by T₁ measurement (Fig. 3B). The inhibitory effect was consistent with the results of Congo red assay and TEM (Fig. 2). On the other hand, the time-dependent increases of T₁ were observed in Gd-DO3A-Chal and Gd-DO3A-Cur. This might be because these two probes were buried in the hydrophobic pocket as Aβ fibril grew up and fewer water molecules permitted access to the Gd ions. It is also possible that these probes have lower binding affinity, especially for matured fibril, and require higher concentrations to produce significant T₁ changes.²⁶ These probe did not produce the significant ΔT₁ between monomer and fibril samples (Fig. 3B and S3, ESI^†), although they showed little inhibition in Congo red assay and TEM (Fig. 2).Open in a separate windowFig. 3(A) Experimental design of T₁-based detection of Aβ fibrillation and inhibition by using the Gd probes. (B) T₁ changes of the Gd probe solutions with pre-incubated fibrils and monomers in PBS at pH 7.4 (mean ± SEM, n = 3). [Gd] = 10 μM, [Aβ] = 20 μM.The feasibility of the Gd probes was further evaluated by in vitro MRI measurement using a 1 tesla scanner. The T₁-weighted images showed that Gd-DO3A-Comp.B produced slight T₁ signal increases with Aβ monomers for 2 and 24 h (Fig. 4A and B). More significant signal increases were observed in the Gd-DO3A-Comp.B with Aβ fibril pre-incubated for 24 h (Fig. 4C). In contrast, Gd-DO3A-Chal and Gd-DO3A-Cur did not show significant signal changes in the presence of Aβ monomers or fibrils (Fig. 4A–C). These results were mostly consistent with the T₁ profile measured by NMR (Fig. 3). Compared to the previously reported Gd-DO3A-Chal that required 100 μM of the probe concentration to detect the equimolar Aβ,¹⁹ Gd-DO3A-Comp.B could detect five-times lower concentration of Aβ (20 μM) with ten-times lower probe concentration (10 μM). Therefore, Gd-DO3A-Comp.B could be promising to further develop highly sensitive diagnostic MRI contrast agents of AD.Open in a separate windowFig. 4 T ₁-weighted images of the Gd probe solutions in the presence of monomeric Aβ at 2 h incubation (A), monomeric Aβ at 24 h incubation (B), and Aβ fibrils pre-incubated for 24 h (C). Incubation was conducted in PBS at pH 7.4.In conclusion, we synthesized the curcumin-based Gd probes which enabled the detection and inhibition of Aβ fibril formation. Gd-DO3A-Comp.B allowed for the highly sensitive detection of Aβ fibril by the T₁ measurement. Moreover, the inhibitory activity could be estimated by T₁ measurement, because Gd-DO3A-Comp.B decreased T₁ depending on the growth stage of Aβ fibril formation. Such unique modality would be useful not only for the diagnostics but also for the direct evaluation of the therapeutic efficacy in vivo. For the future application, it would be important to combine with BBB penetration methods targeting the brain such as transient opening of the BBB using focused ultrasound or mannitol injection.^27,28     

Novel stereoselective syntheses of N-octyl-β-valienamine (NOV) and N-octyl-4-epi-β-valienamine (NOEV) from (−)-shikimic acid

N-Octyl-β-valienamine (NOV) 1 and N-octyl-4-epi-β-valienamine (NOEV) 2 are potent chemical chaperone drug candidates for the therapy of lysosomal storage disorders. Novel stereoselective syntheses of NOV 1 and NOEV 2 starting from naturally abundant (−)-shikimic acid are described in this article. The common key intermediate compound 5 was first synthesized from readily available (−)-shikimic acid via 9 steps in 50% yield. Compound 5 was then converted to NOV 1via 5 steps in 61% yield, and it was also converted to NOEV 2via 8 steps in 38% yield. In summary, NOV 1 was synthesized via 14 steps in 31% overall yield; and NOEV 2 was synthesized via 17 steps in 19% overall yield.

Novel stereoselective syntheses of N-octyl-β-valienamine (NOV) 1 and N-octyl-4-epi-β-valienamine (NOEV) 2 starting from naturally abundant (−)-shikimic acid are described in this article.     

Visible light-induced oxidative α-hydroxylation of β-dicarbonyl compounds catalyzed by ethylenediamine–copper(ii)

We have developed an efficient oxidative α-hydroxylation of β-keto esters with firstly using the structurally simple ethylenediamine–copper(ii) as a catalyst for β-keto esters activation and using visible light as the driving force for generating more active singlet oxygen (¹O₂) from triplet state oxygen (³O₂) in the air, providing a series of α-hydroxy β-keto esters in excellent yields (up to 99%) under extremely low photosensitizer loading (0.01 mol%) and catalyst loading (1 mol%) within a short time. Moreover, the gram-scale synthesis showed the practical utility of this protocol.

An efficient visible light-induced oxidative α-hydroxylation of β-dicarbonyl compounds has been developed using structurally simple ethylenediamine–copper(ii) as a catalyst.     

Hemoglobin Hasharon (α247 his(CD5)β2): a hemoglobin found in low concentration

Hemoglobin Hasharon (alpha(2) (47 his)(CD5)beta(2)) was found to comprise only 16-19% of hemolysates of carriers. These heterozygotes appeared to have mild, compensated, hemolytic anemia. Hb Hasharon was more heat-labile than hemoglobins A, S, or C. Its specific activity was higher than that of Hb A after administration of (59)Fe to two carriers. When hemoglobin synthesis by bone marrow cells was studied in vitro, about 18% of incorporated leucine appeared in the Hb Hasharon fraction. It is suggested that Hb Hasharon is unstable in vivo, and that mild hemolytic anemia and a relatively small decrease in its concentration in hemolysates result from its denaturation within red cells. Decreased synthesis, which appears to be the major cause of the small amount of abnormal hemoglobin, may protect heterozygotes from clinically significant hemolytic anemia.     

Hemoglobin Philly (β35 tyrosine→phenylalanine): studies in the molecular pathology of hemoglobin

An abnormal unstable hemoglobin, hemoglobin Philly, was found in three members of a family, each of whom had evidence of a chronic hemolytic state. The presence of the mutant protein was suggested by the rapid appearance of inclusion bodies upon incubation of erythrocytes with brilliant cresyl blue and by the increased heat precipitability of the hemoglobin. However, no abnormal hemoglobin could be demonstrated by electrophoresis or column chromatography. Sulfhydryl titration of the hemolysates with p-mercuribenzoate indicated that there was an average of four reactive sulfhydryl groups per hemoglobin molecule instead of the usual two. The total number of hemoglobin sulfhydryl groups was normal; six groups were measured when denatured globin was reacted with 5,5'-dithiobis[2-nitrobenzoic acid]. This indicated that the increased sulfhydryl reactivity was due to an increased availability to p-mercuribenzoate of the usually unreactive hemoglobin cysteines at beta112 and alpha104. After treatment for (1/2) hr with 4-5 moles of p-mercuribenzoate per mole of hemoglobin, electrophoresis revealed that 30-35% of the hemoglobin had been dissociated into alpha- and beta-chains. Normal hemolysates revealed negligible splitting after 72 hr of similar treatment. The alpha- and beta-chains of hemoglobin Philly were separated from the unsplit hemoglobin A by carboxymethyl cellulose chromatography. Fingerprint and amino acid analyses revealed that tyrosine beta35 was replaced by phenylalanine. In hemoglobin Philly there is loss of the normal hydrogen bond between the tyrosine hydroxyl group and the carboxyl group of aspartic acid alpha126 at the alpha(1)beta(1) contact. This shifts the equilibrium from hemoglobin tetramers toward monomers, exposing the beta112 and alpha104 cysteines. In the cell, precipitation of the unstable monomers may contribute to erythrocyte destruction.     

Allelic Exclusion in pTα-deficient Mice: No Evidence for Cell Surface Expression of Two T Cell Receptor (TCR)-β Chains, but Less Efficient Inhibition of Endogeneous Vβ→ (D)Jβ Rearrangements in the Presence of a Functional TCR-β Transgene

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-β chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-β chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-β rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-β rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-α (pTα) chain, a core component of the pre-TCR. Staining of CD3⁺ thymocytes and lymph node cells with antibodies specific for Vβ6 or Vβ8 and a pool of antibodies specific for most other Vβ elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTα-deficient mice expressing a functionally rearranged TCR-β transgene. Interestingly, although the transgenic TCR-β chain significantly influenced thymocyte development even in the absence of pTα, it was not able to inhibit fully endogeneous TCR-β rearrangements either in total thymocytes or in sorted CD25⁺ pre-T cells of pTα^−/− mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.