Versatile Reactivity of MnII Complexes in Reactions with N-Donor Heterocycles: Metamorphosis of Labile Homometallic Pivalates vs. Assembling of Endurable Heterometallic Acetates.

Reaction of 2,2'-bipyridine (2,2'-bipy) or 1,10-phenantroline (phen) with [Mn(Piv)2(EtOH)]n led to the formation of binuclear complexes [Mn2(Piv)4L2] (L = 2,2'-bipy (1), phen (2); Piv- is the anion of pivalic acid). Oxidation of 1 or 2 by air oxygen resulted in the formation of tetranuclear MnII/III complexes [Mn4O2(Piv)6L2] (L = 2,2'-bipy (3), phen (4)). The hexanuclear complex [Mn6(OH)2(Piv)10(pym)4] (5) was formed in the reaction of [Mn(Piv)2(EtOH)]n with pyrimidine (pym), while oxidation of 5 produced the coordination polymer [Mn6O2(Piv)10(pym)2]n (6). Use of pyrazine (pz) instead of pyrimidine led to the 2D-coordination polymer [Mn4(OH)(Piv)7(µ2-pz)2]n (7). Interaction of [Mn(Piv)2(EtOH)]n with FeCl3 resulted in the formation of the hexanuclear complex [MnII4FeIII2O2(Piv)10(MeCN)2(HPiv)2] (8). The reactions of [MnFe2O(OAc)6(H2O)3] with 4,4'-bipyridine (4,4'-bipy) or trans-1,2-(4-pyridyl)ethylene (bpe) led to the formation of 1D-polymers [MnFe2O(OAc)6L2]n·2nDMF, where L = 4,4'-bipy (9·2DMF), bpe (10·2DMF) and [MnFe2O(OAc)6(bpe)(DMF)]n·3.5nDMF (11·3.5DMF). All complexes were characterized by single-crystal X-ray diffraction. Desolvation of 11·3.5DMF led to a collapse of the porous crystal lattice that was confirmed by PXRD and N2 sorption measurements, while alcohol adsorption led to porous structure restoration. Weak antiferromagnetic exchange was found in the case of binuclear MnII complexes (JMn-Mn = -1.03 cm-1 for 1 and 2). According to magnetic data analysis (JMn-Mn = -(2.69 ÷ 0.42) cm-1) and DFT calculations (JMn-Mn = -(6.9 ÷ 0.9) cm-1) weak antiferromagnetic coupling between MnII ions also occurred in the tetranuclear {Mn4(OH)(Piv)7} unit of the 2D polymer 7. In contrast, strong antiferromagnetic coupling was found in oxo-bridged trinuclear fragment {MnFe2O(OAc)6} in 11·3.5DMF (JFe-Fe = -57.8 cm-1, JFe-Mn = -20.12 cm-1).

Supplementation with beta-hydroxy-beta-methylbutyrate impacts glucose homeostasis and increases liver size in trained mice.

ß-hydroxy-ß-methyl butyrate (HMB) is a bioactive metabolite derived from the amino acid leucine, usually applied for muscle mass increase during physical training, as well as for muscle mass maintenance in debilitating chronic diseases. The hypothesis of the present study is that HMB is a safe supplement for muscle mass gain by strength training. Based on this, the objective was to measure changes in body composition, glucose homeostasis and hepatic metabolism of HMB supplemented mice during strength training. Two of four groups of male mice (n = 6/group) underwent an 8-week training period session (climbing stairs) with or without HMB supplementation (190 mg/kgBW per day). We observed lower body mass gain (4.9 ± 0.43% versus 1.2 ± 0.43, p < 0.001) and increased liver mass (40.9 ± 0.9 mg/gBW versus 44.8 ± 1.3, p < 0.001) in the supplemented trained group compared with the non-supplemented groups. The supplemented trained group had an increase in relative adipose tissue mass (12.4 ± 0.63 mg/gBW versus 16.1 ± 0.88, P < 0.01) compared to the non-supplemented untrained group, and an increase in fasting blood glucose (111 ± 4.58 mg/dL versus 122 ± 3.70, P < 0.05) and insulin resistance (3.79 ± 0.19 % glucose decay/min versus 2.45 ± 0.28, P < 0.05) comparing with non-supplemented trained group. Adaptive heart hypertrophy was observed only in the non-supplemented trained group (4.82 ± 0.05 mg/gBW versus 5.12 ± 0.13, P < 0.05). There was a higher hepatic insulin-like growth factor-1 expression (P = 0.002) in supplemented untrained comparing with non-supplemented untrained group. Gene expression of gluconeogenesis regulatory factors was increased by training and reduced by HMB supplementation. These results confirm that HMB supplementation associated with intensive training protocol drives changes in glucose homeostasis and liver metabolism in mice.

Development and Validation of Stability-Indicating HPLC Methods for the Estimation of Lomefloxacin and Balofloxacin Oxidation Process under ACVA, H2O2, or KMnO4 Treatment. Kinetic Evaluation and Identification of Degradation Products by Mass Spectrometry.

The oxidation of lomefloxacin (LOM) and balofloxacin (BAL) under the influence of azo initiator of radical reactions of 4,4'-azobis(4-cyanopentanoic acid) (ACVA) and H2O2 was examined. Oxidation using H2O2 was performed at room temperature while using ACVA at temperatures: 40, 50, 60 °C. Additionally, the oxidation process of BAL under the influence of KMnO4 in an acidic medium was investigated. New stability-indicating HPLC methods were developed in order to evaluate the oxidation process. Chromatographic analysis was carried out using the Kinetex 5u XB-C18 100A column, Phenomenex (Torrance, CA, USA) (250 × 4.6 mm, 5 µm particle size, core shell type). The chromatographic separation was achieved while using isocratic elution and a mobile phase with the composition of 0.05 M phosphate buffer (pH = 3.20 adjusted with o-phosphoric acid) and acetonitrile (87:13 v/v for LOM; 80:20 v/v for BAL). The column was maintained at 30 °C. The methods were validated according to the ICH guidelines, and it was found that they met the acceptance criteria. An oxidation process followed kinetics of the second order reaction. The most probable structures of LOM and BAL degradation products formed were assigned by the UHPLC/MS/MS method.

Synthesis and biological evaluation of novel SIPI-7623 derivatives as farnesoid X receptor (FXR) antagonists.

Most of reported steroidal FXR antagonists are restricted due to low potency. We described the design and synthesis of novel nonsteroidal scaffold SIPI-7623 derivatives as FXR antagonists. The most potent compound A-11 (IC50 = 7.8 ± 1.1 µM) showed better activity compared to SIPI-7623 (IC50 = 40.8 ± 1.7 µM) and guggulsterone (IC50 = 45.9 ± 1.1 µM). Docking of A-11 in FXR's ligand-binding domain was also studied.

Improvement of n-caproic acid production with Ruminococcaceae bacterium CPB6: selection of electron acceptors and carbon sources and optimization of the culture medium.

BACKGROUND: Global energy and resource shortages make it necessary to quest for renewable resources. n-Caproic acid (CA) production based on carboxylate platform by anaerobic fermentation is booming. Recently, a novel Ruminococcaceae bacterium CPB6 is shown to be a potential biotransformation factory for CA production from lactate-containing wastewater. However, little is known about the effects of different electron acceptors (EAs) on the fermentative products of strain CPB6, as well as the optimum medium for CA production. RESULTS: In this study, batch experiments were performed to investigate the fermentative products of strain CPB6 in a lactate medium supplemented with different EAs and sugars. Supplementation of acetate, butyrate and sucrose dramatically increased cell growth and CA production. The addition of propionate or pentanoate resulted in the production of C5 or C7 carboxylic acid, respectively. Further, a Box-Behnken experiment was conducted to optimize the culture medium for CA production. The result indicated that a medium containing 13.30 g/L sucrose, 22.35 g/L lactate and 16.48 g/L butyrate supported high-titer CA production (16.73 g/L) with a maximum productivity of 6.50 g/L/day. CONCLUSIONS: This study demonstrated that strain CPB6 could produce C6-C7 carboxylic acids from lactate (as electron donor) with C2-C5 short-chain carboxylic acids (as EAs), but CA (C6 carboxylic acid) was the most major and potential product. Butyrate and sucrose were the most significant EA and carbon source respectively for CA production from lactate by strain CPB6. High titer of CA can be produced from a synthetic substrate containing sucrose, lactate and butyrate. The work provided significant implications for improving CA production in industry-scale.

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.

Salivary Hydrogen Sulfide Measured with a New Highly Sensitive Self-Immolative Coumarin-Based Fluorescent Probe.

Ample evidence suggests that H2S is an important biological mediator, produced by endogenous enzymes and microbiota. So far, several techniques including colorimetric methods, electrochemical analysis and sulfide precipitation have been developed for H2S detection. These methods provide sensitive detection, however, they are destructive for tissues and require tedious sequences of preparation steps for the analyzed samples. Here, we report synthesis of a new fluorescent probe for H2S detection, 4-methyl-2-oxo-2H-chromen-7-yl 5-azidopentanoate (1). The design of 1 is based on combination of two strategies for H2S detection, i.e., reduction of an azido group to an amine in the presence of H2S and intramolecular lactamization. Finally, we measured salivary H2S concentration in healthy, 18⁻40-year-old volunteers immediately after obtaining specimens. The newly developed self-immolative coumarin-based fluorescence probe (C15H15N3O4) showed high sensitivity to H2S detection in both sodium phosphate buffer at physiological pH and in saliva. Salivary H2S concentration in healthy volunteers was within a range of 1.641⁻7.124 µM.

Novel 4-methyl-2-oxopentanoate reductase involved in synthesis of the Japanese sake flavor, ethyl leucate.

Ethyl-2-hydroxy-4-methylpentanoate (ethyl leucate) contributes to a fruity flavor in Japanese sake. The mold Aspergillus oryzae synthesizes leucate from leucine and then the yeast Saccharomyces cerevisiae produces ethyl leucate from leucate during sake fermentation. Here, we investigated the enzyme involved in leucate synthesis by A. oryzae. The A. oryzae gene/cDNA encoding the enzyme involved in leucate synthesis was identified and expressed in E. coli and A. oryzae host cells. The purified recombinant enzyme belonged to a D-isomer-specific 2-hydroxyacid dehydrogenase family and it NADPH- or NADH-dependently reduced 4-methyl-2-oxopentanate (MOA), a possible intermediate in leucine synthesis, to D-leucate with a preference for NADPH. Thus, we designated this novel enzyme as MOA reductase A (MorA). Furthermore, an A. oryzae strain overexpressing morA produced 125-fold more leucate than the wild-type strain KBN8243. The strain overexpressing MorA produced 6.3-fold more ethyl leucate in the sake than the wild-type strain. These findings suggest that the strain overexpressing morA would help to ferment high-quality sake with an excellent flavor. This is the first study to identify the MOA reductase responsible for producing D-leucate in fungi.

Two New Cinnamyl Isovalerate Derivatives from Sabina gaussenii.

Chemical investigation of the 90% acetone extract of the branches and leaves of Sabina gaussenii led to the isolation of two new cinnamyl isovalerate derivatives (1-2) and eighteen known compounds (3-20). Their structures were determined mainly by means of MS, 1D- and 2D-NMR data, and this is the first time these compounds have been reported from this plant. The biological activity test results indicated that the 90% acetone extract showed cytotoxicity against the human lung adenocarcinoma (A549) cell line (IC50 = 0.98 ± 0.1 µg/mL), compound 6 showed cytotoxicities against human cervical carcinoma (HeLa) (IC50 = 0.4 ± 0.1 µM ) and human gastric carcinoma (BGC-823) (IC50 = 0.9 ± 0.2 µM) cancer cell lines, and compound 19 showed cytotoxicities against HeLa (IC50 = 1.5 ± 0.4 µM), BGC-823 (IC50 = 7.0 ± 0.8 µM ), and A549 (IC50 = 10.6 ± 1.5 µM ) cancer cell lines.

Molecular dynamics studies of modular polyketide synthase ketoreductase stereospecificity.

Ketoreductases (KRs) from modular polyketide synthases (PKSs) can perform stereospecific catalysis, selecting a polyketide with a D- or L-α-methyl substituent for NADPH-mediated reduction. In this report, molecular dynamics (MD) simulations were performed to investigate the interactions that control stereospecificity. We studied the A1-type KR from the second module of the amphotericin PKS (A1), which is known to be stereospecific for a D-α-methyl-substituted diketide substrate (dkD). MD simulations of two ternary complexes comprised of the enzyme, NADPH, and either the correct substrate, dkD, or its enantiomer (dkL) were performed. The coordinates for the A1/NADPH binary complex were obtained from a crystal structure (PDB entry 3MJS), and substrates were modeled in the binding pocket in conformations appropriate for reduction. Simulations were intended to reproduce the initial weak binding of the polyketide substrate to the enzyme. Long (tens of nanoseconds) MD simulations show that the correct substrate is retained in a conformation closer to the reactive configuration. Many short (up to a nanosecond) MD runs starting from the initial structures display evidence that Q364, three residues N-terminal to the catalytic tyrosine, forms a hydrogen bond to the incorrect dkL substrate to yield an unreactive conformation that is more favorable than the reactive configuration. This interaction is not as strong for dkD, as the D-α-methyl substituent is positioned between the glutamine and the reactive site. This result correlates with experimental findings [Zheng, J., et al. (2010) Structure 18, 913-922] in which a Q364H mutant was observed to lose stereospecificity.

Synthesis, Crystal Structure and Biological Activity Screening of Novel N-(α-Bromoacyl)-α-amino Esters Containing Valyl Moiety.

Three novel N-(α-bromoacyl)-α-amino esters: methyl 2-(2-bromo-3-methylbutanamido)pentanoate (1), methyl 2-(2-bromo-3-methylbutanamido)-2-phenylacetate (2) and methyl 2-(2-bromo-3-methylbutanamido)-3-phenylpropanoate (3) were synthesized. Single crystal X-ray diffraction data are reported for compounds 1 and 2. The cytotoxicity, antiinflammatory and antibacterial activity of compounds 1-3 were investigated. Additionally, the physico-chemical properties of studied compounds were calculated and an in silico toxicological study of compounds 1-3 was performed. The low level of cytotoxicity and absence of antibacterial and anti-inflammatory activity of 1-3 in tested concentrations might be a beneficial prerequisite for their incorporation in prodrugs.

Construction and characterization of alginate/calcium ß-hydroxy-ß-methylbutyrate hydrogels: Effect of M/G ratios and calcium ion concentration.

Calcium ß-hydroxy-ß-methylbutyrate (CaHMB), a functional calcium salt, is used to maintain and improve muscle health. Here, a new hydrogel material prepared from alginate (ALG) with three M/G ratios (1:1, 2:1, and 1:2) and CaHMB (0-2 mg/mL) was investigated. CaHMB regulates the formation and properties of ALG hydrogels through chelation and hydrogen bonding. When the M/G ratio was 2:1, the anionic groups of CaHMB containing carboxyl and hydroxyl groups formed hydrogen bonds with the polysaccharide chains, hindering the capture of Ca2+ by the G-residue fragments of ALG, which in turn retarded the gelation process. The noncalcium cross-linked polysaccharide chain structure of ALG and the anionic group of CaHMB also affected the water distribution in the hydrogel, especially when M residue content ≥G residue content. Lower M/G ratios and higher CaHMB concentrations could increase the number of "egg box" crosslinking junctions of calcium alginate, and the microstructure was denser in the gel pores, resulting in a stronger gel strength and more free water bound in the gel matrix. This study provides a theoretical and methodological basis for the design of novel hydrogels by studying the crosslinking features of ALG/CaHMB.

Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B.

BACKGROUND: The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG). RESULTS: In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer. CONCLUSIONS: Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.

Identification and characterization of an inhibitor of trichothecene 3-O-acetyltransferase, TRI101, by the chemical array approach.

Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.

Development and validation of LC-MS/MS method for the estimation of ß-hydroxy-ß-methylbutyrate in rat plasma and its application to pharmacokinetic studies.

A simple, sensitive and specific high-performance liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of ß-hydroxy-ß-methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water-acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C(8) (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30-4600 ng/mL (r > 0.998) for HMB. The intra- and inter-day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench-top, autosampler freeze-thaw cycles and long-term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats.

UV resonance Raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization.

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a ß-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 ß-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 ß-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.

Conformational analysis of green apple flavour: the gas-phase structure of ethyl valerate validated by microwave spectroscopy.

We report on the microwave spectrum of ethyl valerate, C(4)H(9)-COO-C(2)H(5), observed by molecular beam Fourier transform microwave spectroscopy (MB-FTMW). Highly accurate rotational and centrifugal distortion constants of the two most abundant conformers were determined. Their structure was investigated by comparison of the experimental rotational constants with those obtained by quantum chemical calculations. MP2/ 6-311++G(d,p) calculations failed to give a proper description of the observed conformers in the supersonic jet. Therefore, also other basis sets were tried using Hartree-Fock, second-order Møller-Plesset (MP2), and B3LYP methods to predict the observed gas-phase structures of the molecule. The quantum chemical results guided the conformer assignment of the rotational constants, obtained from the microwave experiment. One of the two observed conformers has C(s) symmetry, while the most abundant conformer has C(1) symmetry. The main conformer possesses a strong spectrum with high intensities. Additionally, harmonic frequency calculations at different levels of theory where carried out and a low lying vibration of the entire n-butyl group against the rest of the molecule was identified. The symbiotic interplay of microwave gas-phase investigations and quantum chemical calculations becomes evident in our results.

Interactions of human acetylcholinesterase with phenyl valerate and acetylthiocholine: Thiocholine as an enhancer of phenyl valerate esterase activity.

Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site ("PVsite") were deduced.In this paper, we demonstrate that acetylcholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among substrates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems.

Free acid gel form of ß-hydroxy-ß-methylbutyrate (HMB) improves HMB clearance from plasma in human subjects compared with the calcium HMB salt.

The leucine metabolite, ß-hydroxy-ß-methylbutyrate (HMB), is a nutritional supplement that increases lean muscle and strength with exercise and in disease states. HMB is presently available as the Ca salt (CaHMB). The present study was designed to examine whether HMB in free acid gel form will improve HMB availability to tissues. Two studies were conducted and in each study four males and four females were given three treatments in a randomised, cross-over design. Treatments were CaHMB (gelatin capsule, 1 g), equivalent HMB free acid gel swallowed (FASW) and free acid gel held sublingual for 15 s then swallowed (FASL). Plasma HMB was measured for 3 h following treatment in study 1 and 24 h with urine collection in study 2. In both the studies, the times to peak plasma HMB were 128 (sem 11), 38 (sem 4) and 38 (sem 1) min (P < 0·0001) for CaHMB, FASW and FASL, respectively. The peak concentrations were 131 (sem 6), 249 (sem 14) and 239 (sem 14) µmol/l (P < 0·0001) for CaHMB, FASW and FASL, respectively. The areas under the curve were almost double for FASW and FASL (P < 0·0001). Daily urinary HMB excretion was not significantly increased resulting in more HMB retained (P < 0·003) with FASW and FASL. Half-lives were 3·17 (sem 0·22), 2·50 (sem 0·13) and 2·51 (sem 0·14) h for CaHMB, FASW and FASL, respectively (P < 0·004). Free acid gel resulted in quicker and greater plasma concentrations (+185%) and improved clearance (+25%) of HMB from plasma. In conclusion, HMB free acid gel could improve HMB availability and efficacy to tissues in health and disease.

Synthesis and characterization of PHV-block-mPEG diblock copolymer and its formation of amphiphilic nanoparticles for drug delivery.

Despite the recent research interest in the field of nanoparticles delivery system, their structure modification and transport behavior of various hydrophobic drugs is poorly developed. In this article the synthesis of novel amphiphilic diblock copolymer poly([R]-3-hydroxyvalerate)-block-monomethoxy poly(ethylene glycol) (PHV-block-mPEG) was undertaken by modifying the structure of biodegradable and hydrophobic poly([R]-3-hydroxyvalerate) (PHV) with hydrophilic monomethoxy poly(ethylene glycol) (mPEG). The chemical combination of the two blocks was carried out in the melt using bis(2-ethylhexanoate) tin as transesterification catalyst. The synthesized product was characterized by gel permeation chromatography (GPC), 1H nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) analysis. The block copolymer self-assembled into amphiphilic nanoparticles with a core of hydrophobic PHV and a shell of hydrophilic mPEG in aqueous solution. Characterization of the nanoparticles showed the formation of discrete, spherically shaped nanoparticles with mean particle size of 200 +/- 1 nm and zeta potential of -14 +/- 1 mV. A hydrophobic drug thymoquinone was efficiently incorporated into the core hydrophobic domain of the nanoparticles and its release kinetics was studied in vitro. The amphiphilic PEGylated nanoparticles showed biocompatibility when checked in neuronal hippocampal cells of prenatal rat. Our results suggest that the amphiphilic nanoparticles with core-shell structures are potentially useful to develop novel drug carriers.