Application of cyclodextrinase in non-complexant production of γ-cyclodextrin

The production of γ-cyclodextrin usually includes the utilization of organic complexants. However, the non-complexant production of γ-cyclodextrin is always being explored due to the defects of organic complexants. However, in non-complexant production, the separation of γ-cyclodextrin from α- and β-cyclodextrin is still a challenge. Here, the selective hydrolysis ability of a cyclodextrinase designated PpCD (cyclodextrinase from Palaeococcus pacificus) on α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin was proved. The k_cat/K_m values of PpCD for α-cyclodextrin and β-cyclodextrin were roughly 12-fold and 5-fold higher than that of γ-cyclodextrin. It was proved that PpCD had selective hydrolysis ability and its γ-cyclodextrin purification performance was apparent on various simulated cyclodextrin mixtures with reported proportions derived from different CGTases. Besides, the hydrolysis temperature was optimized and it could be seen that 85°C was appropriate for the production of γ-cyclodextrin. In addition, the production of γ-cyclodextrin was achieved by using PpCD in the γ-CGTase reaction products.     

The enhancement of the oral bioavailability of γ-tocotrienol in mice by γ-cyclodextrin inclusion

Cyclodextrin (CD) is widely used in the pharmaceutical and nutritional fields to form an inclusion complex with lipophilic compounds for the improvement of their aqueous solubility, stability and diffusibility under physiological conditions. In this study, we investigated the effect of the γ-tocotrienol (γT3) inclusion complex with CD on its oral bioavailability. Five-week-old C57BL6 mice were fed a vitamin E-free diet for 28 days, followed by the oral administration of 2.79 mg of γT3-rich fraction (TRF) extracted from rice bran or the equivalent dose (14.5 mg) of a CD inclusion complex with TRF (TRF/CD). The levels of γT3 in sequentially collected plasma were determined by LC-MS/MS. The pharmacokinetic study revealed that the plasma concentrations of γT3 were increased and peaked at 6 or 3 h after the oral administration of TRF or TRF/CD, respectively (C_max values of 7.9±3.3 or 11.4±4.5 μM, respectively). The area under the curve of plasma γT3 concentration also showed a 1.4-fold increase in the group administered with TRF/CD compared with the TRF-only group. Furthermore, the mice that had received the TRF/CD tended to reduce the endotoxin shock induced by injection with lethal amounts of Escherichia coli lipopolysaccharide, compared with the mice that had received TRF alone. Taken together, our results suggest that the CD inclusion improved γT3 bioavailability, resulting in the enhancement of γT3 physiological activity, which would be a useful approach for the nutrition delivery system.     

Selective degradation of β- and γ-cyclodextrin by co-digestion using a CGTase fromBacillus ohbensis and α-glucosidase for efficient α-cyclodextrin recovery

A simple and specific recovery method for α-cyclodextrin (α-CD) was developed by employing co-digestion of CD reaction mixtures with CGTase fromBacillus ohbensis and α-glucosidase. The combination of CGTase fromB. ohbensis and α-glucosidase, such as α-amylase, β-amylase, or glucoamylase was examined for the selective degradation of β-and γ-CD in the CD reaction mixture formed by CGTase fromB. macerans. The co-digestion of the CD mixture with Taka-amylase and the CGTase resulted in α-CD and maltodextrins, the combination with β-amylase resulted in α-CD and maltose, and that with glucoamylase resulted in α-CD and glucose. The conditions of selective degradation of β- and γ-CD by co-digestion with the CGTase and glucoamylase were optimized as follows: the incubation pH, 5.5; incubation temperature, 50°C; CGTase concentration, 15 u/g of substrate; glucoamylase, 10 u/g of substrate; substrate concentration, 10% (w/v); the incubation time was fixed for 18 hr from the stand point of operation convenience. Most part of the content was presented in poster session at the 7th International Cyclodextrin Symposium, Tokyo, April 1994.     

The effect of caseinate on inclusion complexes of γ-cyclodextrin for oxidative stabilization of fish oil

In this study, we investigated that when γ-cyclodextrin (γ-CD) forms the inclusion complexes for fish oils, the oxidation stability and retardation of odor can be effectively enhanced through the addition of sodium caseinate (SC). In order to evaluate the oxidative stability and the odor-masking effect, the inclusion complexes of γ-CD and SC incorporating fish oils at various ratios were thermally-oxidized for 5 days at a storage temperature of 55°C. The use of SC on the inclusion complexes of γ-CD could improve the oxidative stability of fish oils and the masking effect of the odor. When the inclusion complexes for fish oils were composed with 80% of γ-CD and 20% of SC, the improvement effect of the oxidative stability was better compared with the complexes of other ratios. Therefore, the use of γ-CD and SC can potentially protecting sensitive ingredients such as EPA and DHA in the food industry.     

Characterization of the mRNAs for α-, β- and γ-actin

The mRNAs for α-, β- and γ-actin have been characterized with respect to molecular weight and poly(A) content. Polyacrylamide gel electrophoresis under denaturing conditions shows that the mRNA for α-actin (muscle-specific actin) is approximately 4.6 × 10⁵ daltons in size, and that the mRNAs for β- and γ-actin (nonmuscle actins) are much larger, approximately 6.6 × 10⁵ daltons in size. We therefore calculate that the noncoding regions of the β- and γ-actin mRNAs contain about 800 nucleotides. This is in marked contrast to the noncoding regions of α-actin mRNA which contain only about 180 nucleotides. During electrophoresis in high-resolution nondenaturing gels, the β-actin mRNA migrates slightly slower than the γ-actin mRNA. This indicates either that β-actin mRNA is about 100 nucleotides longer than γ-actin mRNA, or that these mRNAs differ in secondary structure. Fractionation of actin mRNA on the basis of poly(A) content shows that a substantial portion of the β-actin mRNA, but very little of the α- or γ-actin mRNAs, fails to bind to oligo(dT)-cellulose. Much of this poly(A)-deficient β-actin mRNA, however, does bind to poly(U)-Sepharose, a substrate with higher affinity for short poly(A) sequences. This indicates that many of these β-actin mRNA molecules are polyadenylated, but that they have unusually short poly(A) tails. The finding that β- and γ-actins are translated from mRNAs of different electrophoretic mobility and different poly(A) content strongly suggests that these two closely related proteins are products of different genes.     

Conjugation of oligosaccharides by reductive amination to amine modified chondroitin oligomer and γ-cyclodextrin

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell–cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.     

The Construction of γ-TIP Prokaryotic Expression Vector and Preparation of the γ- TIP Antibody

A prokaryotic expression vector, pGEX-TIP, was constructed from Arabidopsis thaliana (L.) Heynh. Employing PCR, 205 bp fragment near 3' end of γ-TIP cDNA, which has specific aquaporin activity, was amplified and cloned into pGEX-KG. Restriction endonuclease analysis and sequencing confirmed the correct construction, and 0.4 mmoL/L IPTC can induce high expression of GST-TIP fused protein which was about 50% in total of E. coli proteins. The IPTG induced E. coli was collected and ]ysed by supersonic treatment. The fusion protein was mainly recovered as an inclusion body. The expressed GST-TIP was purified by SDS-PAGE according to their molecular weight, which was about 32 kD. The purified protein was used to immune rabbits directly or was electrophoretically eluted before it was used for immunization. The highly qualified antibody for GST-TIP was obtained, which provides a very useful protein probe for the research on localization and function of aquaporins.     

Interplay between α-, β-, and γ-Secretases Determines Biphasic Amyloid-β Protein Level in the Presence of a γ-Secretase Inhibitor

Amyloid-β (Aβ) is produced by the consecutive cleavage of amyloid precursor protein (APP) first by β-secretase, generating C99, and then by γ-secretase. APP is also cleaved by α-secretase. It is hypothesized that reducing the production of Aβ in the brain may slow the progression of Alzheimer disease. Therefore, different γ-secretase inhibitors have been developed to reduce Aβ production. Paradoxically, it has been shown that low to moderate inhibitor concentrations cause a rise in Aβ production in different cell lines, in different animal models, and also in humans. A mechanistic understanding of the Aβ rise remains elusive. Here, a minimal mathematical model has been developed that quantitatively describes the Aβ dynamics in cell lines that exhibit the rise as well as in cell lines that do not. The model includes steps of APP processing through both the so-called amyloidogenic pathway and the so-called non-amyloidogenic pathway. It is shown that the cross-talk between these two pathways accounts for the increase in Aβ production in response to inhibitor, i.e. an increase in C99 will inhibit the non-amyloidogenic pathway, redirecting APP to be cleaved by β-secretase, leading to an additional increase in C99 that overcomes the loss in γ-secretase activity. With a minor extension, the model also describes plasma Aβ profiles observed in humans upon dosing with a γ-secretase inhibitor. In conclusion, this mechanistic model rationalizes a series of experimental results that spans from in vitro to in vivo and to humans. This has important implications for the development of drugs targeting Aβ production in Alzheimer disease.     

Binding of Sulfamethazine to β-cyclodextrin and Methyl-β-cyclodextrin

β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) complexes with sulfamethazine (SMT) were prepared and characterized by different experimental techniques, and the effects of βCD and MβCD on drug solubility were assessed via phase-solubility analysis. The phase-solubility diagram for the drug showed an increase in water solubility, with the following affinity constants calculated: 40.4 ± 0.4 (pH 2.0) and 29.4 ± 0.4 (pH 8.0) M⁻¹ with βCD and 56 ± 1 (water), 39 ± 3 (pH 2.0) and 39 ± 5 (pH 8.0) M⁻¹ with MβCD. According to ¹H NMR and 2D NMR spectroscopy, the complexation mode involved the aromatic ring of SMT included in the MβCD cavity. The complexes obtained in solid state by freeze drying were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and thermal analysis. The amorphous complexes obtained in this study may be useful in the preparation of pharmaceutical dosage forms of SMT.     

Nanopore sensing of γ-cyclodextrin induced host-guest interaction to reverse the binding of perfluorooctanoic acid to human serum albumin

Perfluorooctanoic acid (PFOA) has been one of the most common perfluorochemicals, which are globally pervasive contaminants that are persistent, bioaccumulative, toxic, and have adverse impacts on human health. The highest concentration of PFOA occurs in the blood, where it strongly binds to human serum albumins (HSA). Thus, a method to reverse the HSA-PFOA binding is critical to help facilitate the faster elimination of PFOA from the body to minimize its toxicological effects. Inspired by the remediation effect of cyclodextrin (CD) to PFOA through host-guest interactions, herein, by elucidating inter-molecular interactions using a nanopore sensor, we demonstrated in vitro reversal of the binding of PFOA to HSA using γ-cyclodextrin (γ-CD). The competition behavior for the complexation of PFOA between HSA and γ-CD was discussed in combination with in situ nanopore current recording and nuclear magnetic resonance (NMR) characterization. The present work not only demonstrates the potential therapeutic application of γ-CD for PFOA removal from human blood, but also provides an emerging method for investigating interactions between organic compounds and proteins.     

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with K_Is in the range of 97.5?nM – 7.26?µM and 52.5?nM – 1.81?µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (K_Is of 4.75 – 8.87?µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (K_Is?>?10?µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (K_Is of 59.8?nM – 6.42?µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (K_Is of 33.3?nM – 8.74?µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (K_Is?>?10?µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.     

Synthesis of γ-cyclodextrin/chitosan composites for the efficient removal of Cd(II) from aqueous solution

The synthesis of chitosan-graft-γ-cyclodextrin (Ch-g-γ-CD) using persulfate/ascorbic acid redox system was done and characterized by FTIR, XRD, TGA and SEM/EDX. The optimum yield of the copolymer was obtained using 16 × 10⁻³ M γ-cyclodextrins (γ-CD), 2.8 × 10⁻² M ascorbic acid (AA), 1.8 × 10⁻² M K₂S₂O₈ and 0.1 g chitosan in 25 mL of 2% aqueous formic acid at 45 ± 0.2 °C. The highest percent grafting samples were evaluated for cadmium metal ion (Cd(II)) removal from the aqueous solutions where the sorption capacities were found proportional to the grafting extent. The sorption was pH and concentration dependent where, pH = 8.5 was found to be the optimum value. The adsorption data were modeled using Langmuir and Freundlich isotherms. The equilibrium data followed the Langmuir isotherm model with maximum sorption capacity of 833.33 mg/g. The influence of electrolytes, sodium chloride (NaCl) and sodium sulphate (Na₂SO₄) on Cd(II) uptake was also studied. Desorption of the cadmium loaded Ch-g-γ-CD was accomplished with 0.01 N H₂SO₄. The adsorbent exhibited high reusability and could be successfully recycled for nine cycles where in the ninth cycle 27% adsorption was feasible.     

Kinetic study of the quenching reaction of singlet oxygen by α-, β-, γ-, δ-tocotrienols,and palm oil and soybean extracts in solution

Measurements of the singlet oxygen (¹O₂) quenching rates (k_Q (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, β-, γ-, and δ-tocopherols and -tocotrienols (-Tocs and -Toc-3s)), two vitamin E metabolites (α- and γ-carboxyethyl-6-hydroxychroman), and trolox) in ethanol/chloroform/D₂O (50:50:1, v/v/v) and ethanol solutions at 35?°C. Similar measurements were performed for five palm oil extracts 1–5 and one soybean extract 6, which included different concentrations of Tocs, Toc-3s, and carotenoids. Furthermore, the concentrations (wt%) of Tocs, Toc-3s, and carotenoids included in extracts 1–6 were determined. From the results, it has been clarified that the ¹O₂-quenching rates (k_Q (S)) (that is, the relative SOAC value) obtained for extracts 1–6 may be explained as the sum of the product {Σ k_Q^AO-i (S) [AO-i]/100} of the rate constant (k_Q^AO-i (S)) and the concentration ([AO-i]/100) of AO-i (Tocs, Toc-3s, and carotenoid) included.     

The metabolism of γ-2,3,4,5,6-pentachlorocyclohex-1-ene and γ-hexachlorocyclohexane in rats

1. After intraperitoneal administration, gamma-hexachlorocyclohexane (Gammexane) and gamma-2,3,4,5,6-pentachlorocyclohex-1-ene were converted by rats into 2,3,5- and 2,4,5-trichlorophenol, which were excreted as free phenols and as sulphuric acid and glucuronic acid conjugates. 2. Derivatives of 2,4,5-trichlorophenol and 2,4,5-trichlorophenyl glucosiduronic acid and 2,4-dichlorophenylmercapturic acid were isolated from the urine as metabolites of gamma-2,3,4,5,6-pentachlorocyclohex-1-ene. 3. The phenolic metabolites of gamma-hexachlorocyclohexane and gamma-2,3,4,5,6-pentachlorocyclohex-1-ene isolated from urine were similar to those of 1,2,4-trichlorobenzene, which indicates that the two latter compounds are intermediates in gamma-hexachlorocyclohexane metabolism in rats.     

Physicochemical characterization of α-, β-, and γ-cyclodextrins bioesterified with decanoate chains used as building blocks of colloidal nanoparticles

Nanoparticles of amphiphilic α-, β-, and γ-cyclodextrins were obtained by formulation of cyclodextrins enzymatically transesterified with vinyl decanoate. The product of this synthesis is a mixture of bioesterified cyclodextrins with various degrees of substitution (DS) presenting for a same DS different regio-isomers. In a first step, the efficiency of a MALDI-TOF procedure to characterize the average molecular weight of the derivative bulk mixture was demonstrated by comparing the results with those obtained from complementary NMR and HPLC techniques. In a second step, the ultrastructure of nanoparticles prepared from three different batches of synthesis was investigated and correlated with the average molecular weight and DS of the parent derivative.     

Stereoselection in the binding of Ln DOTA to γ-cyclodextrin: A near infrared circular dichroism study

The formation of an inclusion complex between γ-cyclodextrin and Ln DOTA has been reported in the literature. All Ln DOTA complexes in solution give rise to a network of equilibria between different stereoisomers, which are pairs of enantiomers. By means of near infrared circular dichroism of the Yb derivative, we demonstrate that upon the formation of the host-guest complex, there is a complete stereoselection and that only the Λ(δδδδ) binds.     

Cross-linked hydroxypropyl-β-cyclodextrin and γ-cyclodextrin nanogels for drug delivery: Physicochemical and loading/release properties

Due to their size and high surface-to-volume ratio, nanogels can give some unique drug delivery opportunities. A novel technique to prepare cyclodextrin (CD) nanogels, in which the cross-linking takes place simultaneously with an emulsification/solvent evaporation process, has been implemented. The aqueous phase consisted of γ-cyclodextrin (γCD) or hydroxypropyl-β-cyclodextrin (HPβCD) at a fix concentration of 20% (w/w) with or without hydroxypropyl methylcellulose (HPMC) or agar at various concentrations. The incorporation of the cross-linking agent, ethyleneglycol diglycidyl ether (EGDE), was essential for the nanogel formation. By contrast, nanogels could be formed in the absence of surfactant such as Span 80, which can be attributed to the emulsion stabilizing effect of CDs by forming inclusion complexes with the organic solvent at the interface. Gas chromatography-mass spectrometry (GC-MS) analysis of the nanogels confirmed that dichloromethane levels were below the safety limit and, therefore, that these conditions of the organic solvent evaporation (60 °C for 180 min) led to nanogels that satisfy residual solvent requirements. Infrared analysis (IR), transmission electron microscopy (TEM) and dynamic light scattering (DLS) provided information about the cross-linking degree, the size and the size distribution of the nanogels. The ability of the nanogels to host a molecule that can form inclusion complexes and to sustain its release was tested using 3-methylbenzoic acid (3-MBA) as a probe with a high affinity for both β-cyclodextrin (βCD) and γCD. Permeability tests confirmed that 3-MBA was indeed taken up by the nanogels and then slowly released.     

Identification of mouse selenomethionine α, γ-elimination enzyme

The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH₃SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination reaction of l-cystathionine by the enzymological research. The K _m value for the catalysis of l-cystathionine was 0.81 mM and V _max was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH₃SeH by its α,γ-elimination reaction.