One site fits both: A model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme

R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-P_i (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.     

An effective method to use ionic liquids as reaction media for asymmetric reduction by Geotrichum candidum

An effective method was developed to use an enzyme in ionic liquids; the asymmetric reduction of ketones by Geotrichum candidum in ionic liquids proceeded smoothly with excellent enantioselectivity when the cell was immobilized on water-absorbing polymer containing water, while the reaction without the polymer did not proceed.     

用胆甾型苯并—15—冠—5气相色谱固定相分离乳酸和乳酸乙酯光学异构体

胆甾型苯并－１５－冠－５（简称ＣＨ－Ｂ－１５－Ｃ－５）在不同温度范围内的相态不同，在同一温 度范围内因处理方式不同而物性亦相去甚远，用其作成的ＧＣ固定相兼具气固色谱（ＧＳＣ）、 普通气液色谱（ＧＬＣ）和冠醚液晶气液色谱（ＣＬ－ＧＬＣ）多种功能。用该固定相在不同条件下对 乳酸和乳酸乙酯光学异构体混合物的分高效果进行了比较研究，并探讨了手性拆分机理。     

Growth inhibition in animal cell culture

Eight independent cell lines accumulated ammonia in culture to concentrations between 1.3 and 2.9 mM. The growth inhibition of such concentrations of ammonium chloride when added to culture medium was variable. The cell lines tested could be divided into 3 groups depending on their growth response to 2 mM added NH4Cl. In the first group (293, HDF, Vero, and PQXB1/2) little (less than 14%) or no growth inhibition occurred. In the second group (McCoy and MDCK) a reduction in final cell yield of 50-60% was observed. The third group (HeLa and BHK) was most sensitive to the effects of NH4Cl with growth inhibition (greater than 75%) compared to controls. The growth inhibitory effect of added lactate up to 20 mM was negligible (less than 10%) for 3 cell lines, although one cell line (PQXB1/2) showed greater sensitivity. The interactive effects of ammonia and lactate were determined in a matrix experiment. At lactate (greater than 12 mM) and ammonia (1-4 mM), the growth inhibitory effects of the two components were synergistic. However, at low concentrations of lactate (less than 12 mM) the toxic effect of ammonia was reduced. A proposed mechanism for the sparing effect of lactate on ammonia toxicity is discussed. This may have importance in developing strategies for the optimal growth of ammonia-sensitive cell lines.     

Computer simulation of the steady state currents at enzyme doped carbon paste electrodes

The plate-gap model of porous enzyme doped electrode has been proposed and analyzed. It was suggested that reaction diffusion conditions in pores of bulk electrode resemble particular conditions in thin gap between parallel conducting plates. The model is based on the diffusion equations containing a nonlinear term related to the Michaelis–Menten kinetic of the enzymatic reaction inside gap. Steady state current was calculated for the wide range of given parameters and substrate concentrations. All dependences of current on substrate concentration were approximated by hyperbolas in order to obtain “apparent” parameters (maximal currents and apparent Michaelis constants) of modelled biosensors. Simple approximate relationships between given and apparent parameters were derived. The applicability of theoretical plate-gap model was tested for the case of carbon paste electrodes which were doped with PQQ – dependent glucose dehydrogenase. It was found, that soluble glucose dehydrogenase based biosensors exhibit characteristic features of the theoretical plate-gap biosensors.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

Novel aromatic carboxamides from dehydroabietylamine as potential fungicides: Design,synthesis and antifungal evaluation

The present study was carried out to design and synthesize a number of novel aromatic carboxamide derivatives of dehydroabietylamine. The preliminary antifungal assay indicated that most of title compounds displayed moderate to good antifungal activity toward the six fungal strains in vitro. Compounds 3i, 3q, 4b and 4d showed significant antifungal activity against Sclerotinia sclerotiorum, with EC₅₀ values ranging from 0.067 ～ 0.393 mg/L. Compounds 3i, 4b and 4d also showed pronounced mycelial growth inhibition activities against B. cinerea and A. solani. Furthermore, in the in vivo assay, compound 4b exhibited brilliant protective activity against S. sclerotiorum-infected rape leaves. Meanwhile, the in vivo bioassay on tomato plants infected by B. cinerea showed that compound 3i and 4d displayed excellent protective activity at 200 mg/L, which were near to boscalid. Primary mechanistic study revealed that 4b could inhibit sclerotia formation as well as reduce the exopolysaccharide level. SEM and TEM analysis indicated that 4b possessed a strong ability to destroy the surface morphology of mycelia, cell structure and seriously interfere with the growth of the fungal pathogen. In addition, 4b exhibited good inhibitory activity (IC₅₀ = 23.3 ± 1.6 μM) toward succinate dehydrogenase (SDH). Molecular modeling study confirmed the binding modes between compound 4b and SDH. The above antifungal results and fungicidal mechanism study revealed that this class of dehydroabietylamine derivatives could be potential SDH inhibitors and lead compounds for novel fungicides development.     

难溶杂多酸盐催化合成乳酸正丁酯

合成了硅钨三乙醇胺盐和磷钨酸三乙醇胺盐，并以它们为催化剂，合成了乳酸正丁酯。并对硅钨酸三乙醇胺盐催化合成乳酸正丁的工艺条件进行了研究。该催化剂易回收，可重复使用5次以上。     

DL-半胱氨酸对CCL_4肝中毒小白鼠LDH和SOD活性的影响

小白鼠口服花生油溶四氯化碳（ＣＣＬ４）１３．９５ｇ／ｋｇ（公斤体重）至３６ｈ诱发肝中毒和口服ＣＣＬ４前１０～２０ｍｉｎ于腹腔注入ＤＬ－半胱氨酸１００ｍｇ／ｋｇ．用凝胶圆盘电泳法显示肝中毒和中毒前腹腔注入ＤＬ－半胱氨酸小白鼠的血清乳酸脱氢酶（ＬＤＨ）同工酶和肝匀浆超氧化物歧化酶（ＳＯＤ）的谱带变化．结果表明，中毒鼠血清呈现以ＬＤＨ５带占优势的肝损伤ＬＤＨ同工酶谱；中毒前腹腔注入ＤＬ－半胱氨酸的小白鼠，血清ＬＤＨ同工酶谱ＬＤＨ５显著缩减，其它同工酶带几乎未显现．ＣＣＬ４中毒的肝匀浆ＳＯＤ谱带与正常鼠比较明显变宽或呈现两条同工酶带；中毒前腹注ＤＬ－半胱氨酸的小鼠ＳＯＤ带与正常鼠略同，但有的仍呈现两条微弱的同工酶带．谱带的这些变化表明，ＣＣＬ４诱发肝细胞损伤可能与自由基生成和脂质过氧化有关，ＤＬ－半胱氨酸则可能具有阻断肝损伤的作用；ＣＣＬ４肝中毒在诱发自由基生成的同时又刺激了肝细胞ＳＯＤ活性增强     

磁场对固定化乳酸脱氨酶催化活性的影响

采用流通池光纤荧光光度法，对固定化乳酸脱氢酶（ＬＤＨ）在静磁场作用下的催化活性进行了探讨．发现在０．０８４～０．２３０Ｔ磁场作用下ＬＤＨ的催化活性明显降低，其降低值随磁场强度增强而增大，且与磁场作用的时间和ｐＨ值有关，而在２５～４０℃范围内，温度则无显著影响。