Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [³H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [³H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [³H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [³H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (²²Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of ²²Na in light but not in dark.     

Enantioseparation of Mandelic Acid Enantiomers With Magnetic Nano‐Sorbent Modified by a Chiral Selector

In this study, R(+)‐α‐methylbenzylamine‐modified magnetic chiral sorbent was synthesized and assessed as a new enantioselective solid phase sorbent for separation of mandelic acid enantiomers from aqueous solutions. The chemical structures and magnetic properties of the new sorbent were characterized by vibrating sample magnetometry, transmission electron microscopy, Fourier transform infrared spectroscopy, and dynamic light scattering. The effects of different variables such as the initial concentration of racemic mandelic acid, dosage of sorbent, and contact time upon sorption characteristics of mandelic acid enantiomers on magnetic chiral sorbent were investigated. The sorption of mandelic acid enantiomers followed a pseudo‐second‐order reaction and equilibrium experiments were well fitted to a Langmuir isotherm model. The maximum adsorption capacity of racemic mandelic acid on to the magnetic chiral sorbent was found to be 405 mg g^?1. The magnetic chiral sorbent has a greater affinity for (S)‐(+)‐mandelic acid compared to (R)‐(?)‐mandelic acid. The optimum resolution was achieved with 10 mL 30 mM of racemic mandelic acid and 110 mg of magnetic chiral sorbent. The best percent enantiomeric excess values (up to 64%) were obtained by use of a chiralpak AD‐H column. Chirality 27:835–842, 2015. © 2015 Wiley Periodicals, Inc.     

A recombinant β-O-glucosidase from Caldocellum saccharolyticum to hydrolyse desulfo-glucosinolates

Glucosinolates, natural compounds found in Brassicaceae, can easily be transformed into desulfo-glucosinolates by action of Helix pomatia sulfatase. The recombinant -O-glucosidase from Caldocellum saccharolyticum does not catalyse glucosinolate degradation but can hydrolyse desulfo-glucosinolates (thio-d-glucosidic substrates) to produce the corresponding pure nitriles, including valuable homochiral representatives.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

CD3/T-cell receptor coupling to a pertussis and cholera toxin-insensitive G-protein

We have analyzed the effect of CD3/T-cell receptor stimulation on GTP hydrolysis and GTP binding. We show that stimulation of Jurkat, T-cell, membranes with OKT3 results in a 50% increase in GTP hydrolysis which is specifically inhibited by GDP. Pretreatment of the membranes with neither pertussis toxin nor cholera toxin inhibited the GTP hydrolysis. We also show that stimulation with OKT3 increases the binding of GTPγS to Jurkat membranes. These data strongly implicate the involvement of a G-protein in CD3/T-cell receptor signalling.     

Ser203在大鼠脑驱动蛋白水解ATP中重要作用的晶体学分析

鼠脑驱动蛋白（rat brain kinesin）是一种利用水解ATP所释放的能量在微管束上高速并且连续性运动的常规驱动蛋白. 它在神经突触的物质运输中起着重要作用. 研究驱动蛋白是如何将ATP中储藏的化学能转化为机械动能是理解其运动机能的重要课题. 本课题获得了鼠脑驱动蛋白单体与ATP结构类似物AMPPCP形成的复合物晶体结构. 将这个晶体结构与鼠脑驱动蛋白单体-另一种ATP结构类似物AMPPNP形成的复合物晶体结构以及鼠脑驱动蛋白单体-ATP水解产物ADP形成的复合物晶体结构进行相互比较,揭示了活性中心的开关区域I中丝氨酸203可能作为质子的供体,加速了ATP中gamma-磷酸和beta-磷酸的断裂,从而导致ATP的水解.     

Chain-length dependence of the kinetics of the hyaluronan hydrolysis catalyzed by bovine testicular hyaluronidase

Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8x10(2) g mol(-1) to 2.5x10(5) g mol(-1) and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme-substrate complex is maximum for HA molar masses ranging from 3x10(3) to 2x10(4) g mol(-1). Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2x10(4) g mol(-1), are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.     

Enantiomeric impurities in chiral catalysts,auxiliaries, and synthons used in enantioselective syntheses. Part 5

The enantiomeric excess of chiral starting materials is one of the important factors determining the enantiopurity of products in asymmetric synthesis. Fifty‐one commercially available chiral reagents used as building blocks, catalysts, and auxiliaries in various enantioselective syntheses were assayed for their enantiomeric purity. The test results were classified within five impurities level (ie, <0.01%, 0.01%‐0.1%, 0.1%‐1%, 1%‐10%, >10%). Previously from 1998 to 2013, several reports have been published on the enantiomeric composition of more than 300 chiral reagents. This series of papers is necessitated by the fact that new reagents are forthcoming and that the enantiomeric purity of the same reagent can vary from batch to batch and/or from supplier to supplier. This report presents chiral liquid chromatography (LC) and gas chromatography (GC) methods to separate enantiomers of chiral compounds and evaluate their enantiomeric purities. The accurate and efficient LC analysis was done using newly introduced superficially porous particle (SPP 2.7 μm) based chiral stationary phases (TeicoShell, VancoShell, LarihcShell‐P, and NicoShell).     

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC₅₀ and MBRC₅₀) of dextranase were 2 U ml^?1 and 5 U ml^?1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.