Synthesis of adenosylcobinamide 2-chlorophenyl phosphate,a zwitterionic cobinamide phosphate analog of adenosylcobalamin en route to crystallizable cobinamides

The design and implementation of a new, higher yield synthetic method for synthesizing zwitterionic cobinamide phosphates is described. Adenosylcobinamide 2-chlorophenyl phosphate, beta-AdoCbi-PAr -- a 5,6-dimethylbenzimidazole-free adenosylcobalamin analog, where a 2-chlorophenyl group replaces the ribofuranose and 5,6-dimethylbenzimidazole moieties -- is prepared in tens of milligram quantities, quantities sufficient for crystallization and enzyme trials, amounts 100-fold greater than previously available. The use of (31)P NMR spectroscopy to follow reactions directly, the use of control reactions to learn how to reduce reactant water content, and the use of reaction solvents that completely dissolved the corrinoid reactants were crucial for developing this new synthetic route. beta-AdoCbi-PAr was synthesized in 10% overall isolated yield from cyanocobinamide. Cyanocobinamide was converted to cyanocobinamide 2-chlorophenyl phosphate by direct phosphorylation with 2-chlorophenyl phosphodi-(1,2,4-triazolide) in 25% isolated yield and > or = 98% purity. Sodium borohydride reduction of cyanocobinamide 2-chlorophenyl phosphate and reaction with 5'-chloro-5'-deoxy-adenosine produced beta-AdoCbi-PAr in 42% yield and > or = 98% purity. These compounds were characterized by HPLC, (1)H and (31)P NMR, UV-visible spectroscopy, and liquid secondary ionization mass spectroscopy.     

Enantioselective hydrolysis of rasemic naproxen methyl ester with sol–gel encapsulated lipase in the presence of sporopollenin

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, the Candida rugosa lipase (CRL) was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) in the presence and absence of sporopollenin and activated sporopollenin as additive. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of rasemic Naproxen methyl ester that was studied in aqueous buffer solution/isooctane reaction system. The results indicated that the sporopollenin based encapsulated lipase particularly had higher conversion and enantioselectivity compared to the sol–gel free lipase. In this study, excellent enantioselectivity (E > 400) has been noticed for most lipase preparations (E = 166 for the free enzyme) with an ee value ～98% for S-Naproxen. Moreover, (S)-Naproxen was recovered from the reaction mixture with 98% optical purity.     

Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system

The monoethyl esters of the long chain fatty acids (biodiesel) were prepared by alcoholysis of Jatropha oil, a non-edible oil, by a lipase. The process optimization consisted of (a) screening of various commercial lipase preparations, (b) pH tuning, (c) immobilization, (d) varying water content in the reaction media, (e) varying amount of enzyme used, and (f) varying temperature of the reaction. The best yield 98% (w/w) was obtained by using Pseudomonas cepacia lipase immobilized on celite at 50 °C in the presence of 4–5% (w/w) water in 8 h. It was found that yields were not affected if analytical grade alcohol was replaced by commercial grade alcohol. This biocatalyst could be used four times without loss of any activity.     

Measurement of succinylcholine concentration in human plasma by electrospray tandem mass spectrometry

An electrospray mass spectrometric method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine (SUX) is described. An extraction method compatible with direct infusion inlet was developed and leads to an analysis cycle time of 7--8 min instead of 25 min that would be required for HPLC inlet. SUX was extracted from human plasma on C1 solid-phase cartridges and was analyzed using positive ion electrospray tandem mass spectrometry (ESI-MS/MS). SUX plasma concentrations were determined by a stable isotope dilution assay using hexadeuterosuccinylcholine diiodide (SUXd6) as the internal standard. The calibration curve was prepared using the ratio of intensities of the major product ions in the collision-induced dissociation spectrum for known concentration ratios of SUX and SUXd6 in plasma. Calibration curves for the quantification were linear from 25 to 4000 ng/ml. For intraday precision, CV were < or =6% and accuracy ranged from 98 to 103%. For the interday precision, CV were < or =10% and accuracy ranged from 90 to 102%. This method is specific, sensitive, reproducible, and practical in a clinical setting.     

Snake inhibitors of phospholipase A(2) enzymes

Fragment 53--103 of bovine alpha-lactalbumin, prepared by limited peptic digestion of the protein at low pH, is a 51-residue polypeptide chain crosslinked by two disulfide bonds encompassing helix C (residues 86--98) of the native protein. Refolding of the fully reduced fragment (four--SH groups) is expected to lead to three fully oxidized isomers, the native (61--77, 73--91) and the two misfolded species named ribbon (61--91, 73--77) and beads (61--73, 77--91) isomers. The fragment with correct disulfide bonds was formed in approx. 30% yield when refolding was conducted in aqueous solution at neutral pH in the presence of the redox system constituted by reduced and oxidized glutathione. On the other hand, when the reaction was conducted in 30% (v/v) trifluoroethanol (TFE), the oxidative refolding to the native isomer was almost quantitative. To provide an explanation of the beneficial effect of TFE in promoting the correct oxidative folding, the conformational features of the various fragment species were analyzed by far-UV circular dichroism measurements. The fully reduced fragment is largely unfolded in water, but it becomes helical in aqueous TFE. Correctly refolded fragment is produced most when the helical contents of the reduced and oxidized fragment in aqueous TFE are roughly equal. It is proposed that 30% TFE promotes a native-like format of the fragment and thus an efficient and correct pairing of disulfides. Higher concentrations of TFE, instead, promote some non-native helical secondary structure in the fragment species, thus hampering correct folding.     

Preparation and quality evaluation of coenzyme Q10 long-circulating liposomes

The aim of this work was to prepare coenzyme Q10 (CoQ10) long-circulating liposomes, and establish the quality standard to determine the content and entrapment efficiency. CoQ10 long-circulating liposomes were prepared by the film dispersion method, HPLC assay for the determination of CoQ10 was developed. Free drugs and liposomes were separated using the protamine aggregation method and entrapment efficiency was determined. The liposomes were homogeneous and the mean diameter was 166.0 nm, Zeta potential was −22.2 mV. The content and entrapment efficiency of CoQ10 were 98.2% and 93.2% for three batches of liposomes, respectively. The lyophilized form of liposomes prepared by freeze-drying showed stable quality characteristics during storage. The formulation and preparative method can be used to prepare CoQ10 long-circulating liposomes with high entrapment efficiency and high quality, the determination method of drug content and entrapment efficiency were effective and rapid and can be used for quality evaluation of liposomes.     

Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue

A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.     

Preparation of cell walls of group A Streptococcus. Methods of disintegration, isolation and control]

A method for disintegration of cells of group A streptococcus on the Edebeau extrusion press was developed. The level of disintegration was controlled by cell count in stained preparations, Coultier electronic counter and electron microscope. The streptococcus cell disintegration in the Edebeau extrusion press at a temperature of --40 degrees, a pressure of 3200 kg/cm2 and two cycles of the process was completed by 96-98%.     

Evaluation of Alginate/Chitosan nanoparticles as antisense delivery vector: Formulation,optimization and in vitro characterization

Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method through drop wise addition of various concentrations of CaCl₂ to a defined concentration of Sodium Alginate. Then, Chitosan/Antisense solution with a certain N/P ratio was added to the pregel to make the nanoparticles. The effect of such parameters as polymer ratio, CaCl₂/Alginate ratio and N/P ratio on the particle size distribution and loading efficacy was studied. The optimum conditions were 1:1 (w/w) Alginate to Chitosan ratio, 0.2% CaCl₂/Alginate ratio and N/P ratio of 5 at pH 5.3. The resulting nanoparticles had a loading efficacy of 95.6% and average size of 194 nm as confirmed by PCS method and SEM images showed spherical and smooth particles. The zeta potential of optimized nanoparticles prepared by this method was about +30 mV which could result in good stability of nanoparticles during manipulation and storage.     

Chitosan microcapsules loaded with either miconazole nitrate or clotrimazole,prepared via emulsion technique

In this paper, a simple and versatile coacervation technique has been developed by using an ultrasound-assisted oil/water emulsion method for the preparation of antifungal agent-loaded microcapsules. Two types of chitosan microcapsules are successfully prepared. The mean particle size of the chitosan/miconazole nitrate microcapsules is 2.6 μm and that of the chitosan/clotrimazole microcapsules is 4.1 μm. The encapsulation efficiency of the chitosan/miconazole nitrate microcapsules (77.58–96.81%) is relatively higher than that of the chitosan/clotrimazole microcapsules (56.66–93.82%). The in vitro drug release performance of the microcapsules shows that the chitosan/miconazole nitrate microcapsules release about 49.5% of the drug while chitosan/clotrimazole microcapsules release more than 66.1% of the drug after 12 h under a pressure of 5 kg at pH 5.5, which is similar to the pH of human skin. The prepared drug-loaded microcapsules could be applied onto bandages or socks, and will continuously release antifungal drugs in a controlled manner under pressure.     

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65 °C over 6 h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.     

A high-performance liquid chromatography method for the quantification of cysmethynil,an inhibitor of isoprenylcysteine carboxylmethyl transferase,in mouse plasma

Cysmethynil, a newly identified small molecule inhibitor of isoprenylcysteine carboxylmethyl transferase (Icmt) is involved in the post-translational modification of CaaX proteins. Cysmethynil causes cell death in many human cancer cells in vitro, and inhibits tumor growth in the xenograft mouse model in vivo. A HPLC method for the quantification of cysmethynil in mouse plasma was developed and validated. The lower limit of quantification of this method was 0.01 μg/ml. Inter- and intra-day variability ranged from 0.38–8.5% and accuracy was between 86% and 98%. This sensitive method was used to quantify cysmethynil in plasma of mice after intraperitoneal dosing for preliminary pharmacokinetic studies.     

Polyelectrolyte complexes of gum kondagogu and chitosan,as diclofenac carriers

Polyelectrolyte complexes (PEC) of gum kondagogu (GKG) and chitosan were prepared by mixing polymeric solutions of different concentrations (0.02–0.18% w/v). The complex formed were loaded with diclofenac sodium, and the release of the drug was measured in vitro and in vivo, along with the measurement of particle size, zeta potential, complex formation, flow properties, and loading efficiency. Maximum yield of PEC was observed at gum kondagogu concentrations above 80%. The PEC showed lower release of diclofenac sodium in 0.1 N HCl as compared to phosphate buffer (pH 6.8). Increasing the concentration of gum kondagogu in PEC led to an increase in drug release. However, PEC 1:3 (gum kondagogu: chitosan) with higher concentration of chitosan showed 98% release with in 4.5 h, owing to the fact that chitosan has a higher degree of swelling in acidic medium. PEC 5:1 and 3:1 showed a 5.3- and 5.8-fold increase in relative bioavailability compared to the free drug when administered orally to the rats.     

One-step simultaneous purification of three water-soluble constituents in crude extracts from Danshen by adsorption chromatography on oligo-β-cyclodextrin substituted agarose gel media

A new adsorption chromatographic method for the simultaneous purification of Danshensu, Protocatechuic aldehyde and Salvianolic acid B (Lithospermic acid B) from a crude extract from radix of Danshen (Salviae miltiorrhiza) has been developed using oligo-β-cyclodextrin immobilized to Sepharose HP agarose gel media. Sample loads of up to 2.3 mg crude extracts/ml gel gave satisfactory results. The target components were eluted using step-wise isocratic elution with increasing concentrations of acetic acid. The separated fractions were identified by LC–MS. The recoveries of Danshensu, Protocatechuic aldehyde and Salvianolic acid B were 88.3%, 90.2% and 73.6% with purities of 99%, 99%, and 98%, respectively. Isocratic elution in 6% acetic acid gave baseline separation of Danshensu and Protocatechuic aldehyde, whereas 40% acetic acid was required for purification of Salvianolic acid B. Full separation efficiency could be restored by cleaning the column with 0.5 M NaOH followed by distilled water and 30% acetic acid.     

A two-step enzymatic resolution of glycidyl butyrate

A two-step enzymatic resolution process for production of (R)- and (S)-glycidyl butyrate was investigated and the lipases were screened. The first step involved a hydrolysis of (R,S)-glycidyl butyrate catalyzed by porcine pancreatic lipase (S-favored) with an E of 21 for production of (R)-glycidyl butyrate (13.2 mmol, 98% ee, 36% yield) under the optimal conditions (pH 7.4, 30 °C, 30 mg/ml CTAB). Then, the recovered (R)-enriched glycidol (19.8 mmol, 65% ee, 56% yield) was used for transesterification catalyzed by Novozym 435 (R-favored) with an E of 69 to obtain (S)-glycidyl butyrate (15.1 mmol, 98% ee, 42% yield) under the optimum conditions (a_W = 0.24, n-heptane, 80 min).     

Determination of seed viability of eight wild Saudi Arabian species by germination and X-ray tests

Our purpose was to evaluate the usefulness of the germination vs. the X-ray test in determining the initial viability of seeds of eight wild species (Salvia spinosa, Salvia aegyptiaca, Ochradenus baccatus, Ochradenus arabicus, Suaeda aegyptiaca, Suaeda vermiculata, Prosopisfarcta and Panicumturgidum) from Saudi Arabia. Several days were required to determine viability of all eight species via germination tests, while immediate results on filled/viable seeds were obtained with the X-ray test. Seeds of all the species, except Sa.aegyptiaca, showed high viability in both the germination (98–70% at 25/15 °C, 93–66% at 35/25 °C) and X-ray (100–75%) test. Furthermore, there was general agreement between the germination (10% at 25/15 °C and 8% at 35/25 °C) and X-ray (5%) tests that seed viability of Sa.aegyptiaca was very low, and X-ray analysis revealed that this was due to poor embryo development. Seeds of P.farcta have physical dormancy, which was broken by scarification in concentrated sulfuric acid (10 min), and they exhibited high viability in both the germination (98% at 25/15 °C and 93% at 35/25 °C) and X-ray (98%) test. Most of the nongerminated seeds of the eight species except those of Sa.aegyptiaca were alive as judged by the tetrazolium test (TZ). Thus, for the eight species examined, the X-ray test was a good and rapid predictor of seed viability.     

Use of a second-dose calculation algorithm to check dosimetric parameters for the dose distribution of a first-dose calculation algorithm for lung SBRT plans

PurposeTo verify lung stereotactic body radiotherapy (SBRT) plans using a secondary treatment planning system (TPS) as an independent method of verification and to define tolerance levels (TLs) in lung SBRT between the primary and secondary TPSs.MethodsA total of 147 lung SBRT plans calculated using X-ray voxel Monte Carlo (XVMC) were exported from iPlan to Eclipse in DICOM format. Dose distributions were recalculated using the Acuros XB (AXB) and the anisotropic analytical algorithm (AAA), while maintaining monitor units (MUs) and the beam arrangement. Dose to isocenter and dose-volumetric parameters, such as D₂, D₅₀, D₉₅ and D₉₈, were evaluated for each patient. The TLs of all parameters between XVMC and AXB (TL_AXB) and between XVMC and AAA (TL_AAA) were calculated as the mean ± 1.96 standard deviations.ResultsAXB values agreed with XVMC values within 3.5% for all dosimetric parameters in all patients. By contrast, AAA sometimes calculated a 10% higher dose in PTV D₉₅ and D₉₈ than XVMC. The TL_AXB and TL_AAA of the dose to isocenter were −0.3 ± 1.4% and 0.6 ± 2.9%, respectively. Those of D₉₅ were 1.3 ± 1.8% and 1.7 ± 3.6%, respectively.ConclusionsThis study quantitatively demonstrated that the dosimetric performance of AXB is almost equal to that of XVMC, compared with that of AAA. Therefore, AXB is a more appropriate algorithm for an independent verification method for XVMC.     

Effect of trace Ag+ adsorption on degradation of organic dye wastes

By the introduction of Ag⁺, the molecular imprinting technology and photocatalysis technology were associated with each other and the Ag⁺-imprinted biosorbent (Ag-IB) was prepared. Ag-IB could first adsorb Ag⁺ and then degraded Methyl Orange (MO). Firstly the influences of the ionic strength and pH value in solution on adsorption capacity for Ag⁺ were studied, and then the effects of Ag⁺ adsorption capacity on degradability for MO were investigated. The maximal degradation ratio of MO reached over 93% at Ag⁺ adsorption capacity of 78.0 mg/g after 5.0 h. In contrast to MO, Methylene Blue (MB) and Sunset Yellow (SY) were studied and the degradation ratios could be about 70% and 98% at Ag⁺ adsorption capacity of 36.9 mg/g, respectively. And XPS analysis showed that Ag⁺ was reduced to Ag on Ag-IB surface. Furthermore, the mechanism for photocatalytic degradation of MO dye was primarily researched.     

Dosimetric comparison of MRI-based HDR brachytherapy and stereotactic radiotherapy in patients with advanced cervical cancer: A virtual brachytherapy study

AimTo evaluate the treatment plans of 3D image-guided brachytherapy (BT) and stereotactic robotic radiotherapy with online image guidance – CyberKnife (CK) in patients with locally advanced cervix cancer.Methods and materialsTen pairs of plans for patients with locally advanced inoperable cervical cancer were created using MR based 3D brachytherapy and stereotaxis CK. The dose that covers 98% of the target volume (HR CTV D98) was taken as a reference and other parameters were compared.ResultsOf the ten studied cases, the dose from D100 GTV was comparable for both devices, on average, the BT GTV D90 was 10–20% higher than for CK. The HR CTV D90 was higher for CK with an average difference of 10–20%, but only fifteen percent of HR CTV (the peripheral part) received a higher dose from CK, while 85% of the target volume received higher doses from BT. We found a significant organ-sparing effect of CK compared to brachytherapy (20–30% lower doses in 0.1 cm³, 1 cm³, and 2 cm³).ConclusionBT remains to be the best method for dose escalation. Due to the significant organ-sparing effect of CK, patients that are not candidates for BT could benefit from stereotaxis more than from classical external beam radiotherapy.     

Enhanced d-hydantoinase activity performance via immobilized cobalt ion affinity membrane and its kinetic study

Various immobilized metal ions affinity membranes (IMAMs) were prepared from the regenerated cellulose membrane (RC membrane) and chelated with various metal ions such as Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺. The D-hydantoin-hydrolyzing enzyme (DHTase) harboring a poly-His tagged residue was used as a model protein to be immobilized on the prepared IMAMs through the direct metal–protein interaction forces. The adsorption isotherm and the kinetic parameters V_max, K_m,app of DHTase on IMAMs were studied. The cobalt ions chelated IMAM (Co-IMAM) was found to yield the highest specific activity of DHTase. Under the immobilization condition, the cobalt ion chelated amount was 161.4 ± 4.7 μmol/disk with a DHTase activity of 4.1 ± 0.1 U/disk. As compared to the free DHTase, the immobilized DHTase membrane could achieve a broader pH tolerance and higher thermal stability. In addition, 98% of the residual activity could be retained for 7-times repeated use. Only little activity loss was observed within 36-day storage at 4 °C. This is the first report concerning about using cobalt ion as the effective chelated metal ion for simultaneous purification and immobilization operation.