一种费氏丙酸杆菌细胞离位转化生产维生素B_(12)的方法

为建立费氏丙酸杆菌的半连续耦合发酵工艺,克服DMB对维生素B_(12)连续发酵的不利影响,考察了费氏丙酸杆菌菌体细胞离位转化合成维生素B_(12)的可行性,优化了其离位转化工艺,确定了最佳的转化时机、转化体系及DMB添加方式,具体如下:当发酵进行至84 h时,将发酵液离心,收集菌体,然后用离心上清液重悬菌体,配成5倍浓度的菌液,加入终浓度为4.5 mg/L的DMB,于30℃条件下转化48 h,维生素B_(12)的产量达到108.06 mg/L,转化效率为2.26 mg/(Lh)。     

穿山龙多糖的提取、纯化与抗氧化活性研究

采用热水煮提法和乙醇沉淀法从穿山龙中提取出水溶性粗多糖,再经DEAE-Sepharose CL-6B和Sepha-dex G-100柱层析分离纯化得到2个均多糖组分DMA和DMB。利用超氧阴离子实验,羟基阴离子实验和邻苯三酚自氧化实验研究DMA和DMB的抗氧化活性,结果表明DMB有较强的抗氧化活性,应该被作为潜在的抗氧化剂开发利用。     

HPGMR农垦58s光敏感期叶片中阳离子过氧化物酶的变化

以逐垦５８ｓ水稻为材料,用分光光度法和聚丙烯酰胺凝胶电泳,分析了５８ｓ在１０ｈ／ｄ短日照（５８ｓ－ＳＤ）和自然不育长日照（１４．１２ｈ／ｄ左右,５８ｓ－ＮＤ）条件下,育性转换光敏感期,阳离子过氧化物酶活性同工酶的变化。结果表明,在育性转换期阳离子过氧化物酶活性随幼穗发育时期的推进而升高,在ＮＤ条件下,酶活性升高更快。在所测定的２上时期中,收丙烯酰胺凝胶电泳图谱可以况５８ｓ－ＳＤ和５８ｓ－ＮＤ均有５     

β-甘露聚糖酶发酵液絮凝条件的统计学筛选与响应面优化

采用Plackett-Burman(PB)和中心复合设计(Central Composite Design)对影响地衣芽孢杆菌TJ-101发酵生产β-甘露聚糖酶粗发酵液絮凝操作的8个条件进行筛选优化。PB实验设计与统计学分析表明:加水量、阳离子聚丙烯酰胺(C-PAM)和阴离子聚丙烯酰胺(A-PAM)的体积分数是影响酶活收率的3个关键因素。以酶活收率为响应目标,对3因素进行中心复合设计,并经响应面法优化分析得到影响酶活收率的二阶模型,确定了β-甘露聚糖酶发酵液絮凝实验的最优操作条件为:加水量体积分数240.55%,C-PAM体积分数14.13%,A-PAM体积分数16.97%,絮凝后发酵液的酶活收率达70.42%。     

鸡心线粒体肌酸激酶的分离纯化

线粒体肌酸激酶(Mi-CK)的分离纯化采用了制备线粒体、低渗胀破线粒体外膜、磷酸盐抽提出线粒体肌酸激酶后再经硫铵分级、DEAE-Sepharose Fast Flow 阴离子交换层析、FPLC Mono-S 阳离子交换层析的方法,从鸡心中分离出纯度大于99%的Mi-CK,经 SDS 聚丙烯酰胺凝胶电泳和醋酸纤维素薄膜电泳鉴定均为一条带,收率为15.3%,比活力为60U/mg.     

贵州汉族人群HLA-DM基因多态性分析

为了探讨贵州汉族人群HLA-DM基因多态性的分布情况, 采用PCR-RFLP法对125 例贵州汉族人进行HLA-DM基因分型。结果显示, 贵州汉族人群DMA*0101~0103等位基因频率依次是0.720、0.244、0.036, DMB*0101~0104等位基因频率分布依次是0.620、0.156、0.188和0.036; 贵州汉族人群中DMA的基因型以DMA*0101/0101和0101/0102为主, 而DMB的基因型以DMB*0101/0101、0101/0102和0101/0103为主。结果表明, HLA-DM基因多态性具有地区性、民族性的遗传特征。     

水华微囊藻混凝沉降去除过程中铝盐回收及循环使用

以水华微囊藻(Microcystis flos-aquae)为研究对象,对用聚合氯化铝和阳离子聚丙烯酰胺收获后的生物质进行酸洗处理回收铝盐并进行多次循环使用,以降低混凝沉降工艺中吨水处理成本.研究结果表明,总铝盐回收效率随酸洗次数增加而增大随后达到最大,而在较高的盐酸浓度或较高的液-固比条件下可最快达到较高的铝盐回收效...     

TMAO对脾虚高脂血症大鼠脂代谢的影响及香砂六君子汤的干预作用

目的 本研究拟探讨肠道菌群代谢物TMAO对脾虚高脂血症大鼠肝脂质代谢的影响,并进一步探讨香砂六君子汤干预肝脂质代谢紊乱的可能机制。方法 SD大鼠分为：空白对照组(C组);空白对照组+DMB(C+D组),DMB为TMAO抑制剂;脾虚高脂血症组(PG组);脾虚高脂血症+DMB组(PG+D组);脾虚高脂血症+香砂六君子汤组(PG+XS组)。除C组、C+D组外各组构建以劳倦过度和高脂饲料相结合建立脾虚高脂血症模型(造模12周),模型建立后C+D组、PG+D组每天饮用水中给予1%DMB,PG+XS组每天灌胃香砂六君子汤(每天11.34 g生药/kg),其余给予等量生理盐水,4周后进行取材检测。全自动生化仪检测大鼠血脂含量;HE染色、油红O染色观察大鼠肝变化及脂质沉积情况;ELISA法检测大鼠肝FFA变化;试剂盒法检测各组大鼠TG、TC含量;LC-MS法检测大鼠血浆TMAO含量;qRT-PCR法检测大鼠肝PERK、FOXO1、SREBP-2、ABCA1、miR-33 mRNA相对表达水平;Western Blot检测大鼠肝SREBP-2、ABCA1蛋白表达。结果 (1)PG组大鼠血清中TC、TG和...     

桑叶凝集素分离纯化及抑菌活性研究

采用磷酸缓冲液提取桑叶凝集素,经硫酸铵分级沉淀法初步纯化、Cellulose DE-52阴离子柱层析、CMSepharose Fast Flow阳离子柱层析、Sephadex G-75分子筛柱层析对其进行逐级纯化,采用牛津杯法进行抑菌活性测试。结果表明桑叶凝集素经聚丙烯酰胺凝胶电泳检测显示为单一条带,SDS聚丙烯酰胺凝胶电泳检测其亚基分子量为35.95、30.35 KD;该凝集素对供试菌具有较好的抑制作用,对绿色木霉、苹果落叶病菌、西瓜枯萎病菌及烟草黑胫病菌抑菌直径分别为20、30、15、18 mm。桑叶凝集素具有较好的抑菌活性,可为新农药创制领域提供有用的天然产物先导化合物。     

改性瓜尔胶的制备及其絮凝性能的研究

对瓜尔胶进行铵基化阳离子改性,得到一种天然絮凝剂.研究了改性瓜尔胶絮凝剂处理污水的效果,以及pH值、反应温度、粘度、取代度等因素对高岭土悬浮液絮凝效果的影响.结果表明,粘度为3 000～5 000mPa·s,阳离子取代度为13%的改性瓜胶对高岭土悬浮液有较好的絮凝效果,在胶加入量为0.2 mL/L,搅拌速度为500r/min、搅拌时间为15 min等条件下,沉降15 min可达到彻底絮凝和澄清.此外,该絮凝剂基本不受温度和水质pH影响,且絮凝性能明显优于聚丙烯酰胺、硫酸铝以及三氯化铁等絮凝剂,因而在工业废水处理中具有良好的应用前景.     

An Orally Active Allosteric GLP-1 Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke

Diabetes is a major risk factor for the development of stroke. Glucagon-like peptide-1 receptor (GLP-1R) agonists have been in clinical use for the treatment of diabetes and also been reported to be neuroprotective in ischemic stroke. The quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) is an agonist and allosteric modulator of the GLP-1R with the potential to increase the affinity of GLP-1 for its receptor. The aim of this study was to evaluate the neuroprotective effects of DMB on transient focal cerebral ischemia. In cultured cortical neurons, DMB activated the GLP-1R, leading to increased intracellular cAMP levels with an EC₅₀ value about 100 fold that of exendin-4. Pretreatment of neurons with DMB protected against necrotic and apoptotic cell death was induced by oxygen-glucose deprivation (OGD). The neuroprotective effects of DMB were blocked by GLP-1R knockdown with shRNA but not by GLP-1R antagonism. In C57BL/6 mice, DMB was orally administered 30 min prior to middle cerebral artery occlusion (MCAO) surgery. DMB markedly reduced the cerebral infarct size and neurological deficits caused by MCAO and reperfusion. The neuroprotective effects were mediated by activation of the GLP-1R through the cAMP-PKA-CREB signaling pathway. DMB exhibited anti-apoptotic effects by modulating Bcl-2 family members. These results provide evidence that DMB, a small molecular GLP-1R agonist, attenuates transient focal cerebral ischemia injury and inhibits neuronal apoptosis induced by MCAO. Taken together, these data suggest that DMB is a potential neuroprotective agent against cerebral ischemia.     

Demethyleneberberine induces cell cycle arrest and cellular senescence of NSCLC cells via c-Myc/HIF-1α pathway

BackgroundDemethyleneberberine (DMB) is a natural active component of medicinal plant Cortex phellodendri chinensis with favorable bioactivity. However, the role of DMB in suppressing non-small cell lung cancer (NSCLC) remains unknown.PurposeIn this study, we aimed to examine the effect and underlying mechanism of DMB in suppressing NSCLC.MethodsCCK8 assay and colony formation assay were utilized to assess the efficiency of DMB on the viability and colony formation capacity of NSCLC cells. Flow cytometry and β-Galactosidase Staining Kit were utilized to determine the efficiency of DMB on the cell cycle and cellular senescence of NSCLC cells. RT-qPCR and Western blot were used to detect the effect of DMB on cell cycle and cellular senescence related gene and protein expression of NSCLC cells. In vivo tumor model was established to evaluate the anti NSCLC effect of DMB. In addition, RNA-seq analysis was performed to detect the differential gene expression after DMB treatments.ResultsIn this study, we revealed that DMB exhibits efficient inhibitory effect on NSCLC cell proliferation and tumor xenografts growth in vivo. We also demonstrated that DMB could inhibit cell migration by suppressing epithelial-mesenchymal transition (EMT) and trigger cell cycle arrest by down-regulating the expression of cell cycle related genes in NSCLC cells. In addition, DMB treatment efficiently induces cellular senescence of NSCLC cells. From the RNA-seq analysis, we found that DMB accelerates senescence through suppressing HIF-1α expression, which was further elucidated by overexpressing HIF-1α in NSCLC to reduce the inhibitory effect of DMB. Furthermore, we also revealed that DMB decreases the expression of c-Myc, an up-stream protein of HIF-1α.ConclusionsTaken together, we first report that DMB inhibits NSCLC progress through inducing cell cycle arrest and triggering cellular senescence by downregulating c-Myc/HIF-1α pathway.     

A quinoxaline-based compound ameliorates bone loss in ovariectomized mice

DMB (6,7-dichloro-2-methylsulfonyl-3-Ntert-butylaminoquinoxaline) is a quinoxaline-based compound that has been investigated as a glucagon-like peptide-1 receptor (GLP-1R) agonist. To clarify anti-osteoporosis effect of DMB, an osteoporotic mice model was established by ovariectomy (OVX) operation. The OVX mice were given intraperitoneally DMB, exendin-4 (EX-4), or 17β-estradiol (E₂) for two months. Then bone mass and structure, and bone morphometric parameters were examined by micro-CT. Weight gain and food consumption, bone turnover markers, and biomechanical strength of the femur were tested, and bone histomorphometry was analyzed. The food intake and weight gain was obviously reduced by E₂ or EX-4, but not DMB. However, DMB or EX-4 treatment obviously inhibited skeletal deterioration and enhanced bone strength. The improvement involved in the increased osteoblast number and level of bone formation markers, and reduced osteoclasts number and level of bone resorption markers. In addition, DMB was found to stimulate osteoblastogenesis-related marker gene expression. These results demonstrated that DMB ameliorated bone loss mainly via induction of bone formation, which suggests that the small molecule compound might be applied to the management of postmenopausal osteoporosis.     

3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice

Trimethylamine N-oxide (TMAO) is closely related to cardiovascular diseases, particularly heart failure (HF). Recent studies shows that 3,3-dimethyl-1-butanol (DMB) can reduce plasma TMAO levels. However, the role of DMB in overload-induced HF is not well understood. In this research study, we explored the effects and the underlying mechanisms of DMB in overload-induced HF. Aortic banding (AB) surgery was performed in C57BL6/J mice to induce HF, and a subset group of mice underwent a sham operation. After surgery, the mice were fed with a normal diet and given water supplemented with or without 1% DMB for 6 weeks. Cardiac function, plasma TMAO level, cardiac hypertrophy and fibrosis, expression of inflammatory, electrophysiological studies and signaling pathway were analyzed at the sixth week after AB surgery. DMB reduced TMAO levels in overload-induced HF mice. Adverse cardiac structural remodeling, such as cardiac hypertrophy, fibrosis and inflammation, was elevated in overload-induced HF mice. Susceptibility to ventricular arrhythmia also significantly increased in overload-induced HF mice. However, these changes were prevented by DMB treatment. DMB attenuated all of these changes by reducing plasma TMAO levels, hence negatively inhibiting the p65 NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway. DMB plays an important role in attenuating the development of cardiac structural remodeling and electrical remodeling in overload-induced HF mice. This may be attributed to the p65 NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway inhibition.     

Identification of 5,6-dimethylbenzimidazole as the Co alpha ligand of the cobamide synthesized by Salmonella typhimurium. Nutritional characterization of mutants defective in biosynthesis of the imidazole ring.

The Co beta-cyano derivative of the cobamide isolated from Salmonella typhimurium was identified as Co alpha-(alpha-5,6-dimethylbenzimidazolyl)-Co beta-cyanocobamide, indicating that this bacterium synthesizes 5,6-dimethylbenzimidazole (DMB) de novo. We found that mutants deficient in the synthesis of DMB can incorporate benzimidazole without modification to form Co alpha-(alpha-benzimidazolyl)cobamide, a cobamide that is physiologically active. The analysis of the nutritional requirements of mutants deficient in DMB synthesis identified 4,5-dimethylphenylenediamine as a putative intermediate in the synthesis of the imidazole ring of DMB. Our results suggest that the CobII region of the cob operon of S. typhimurium only encodes functions involved in the synthesis of the imidazole ring of DMB.     

Liquid chromatography-mass spectrometric assay for quantitation of the short-chain fatty acid, 2,2-dimethylbutyrate (NSC 741804), in rat plasma

2,2-Dimethylbutyrate (DMB) is a potential treatment for thalassemia and hemoglobinopathies. To facilitate pharmacokinetic evaluation of DMB, we developed an LC-MS assay and quantitated DMB in plasma of rats after an oral dose of 500mg/kg. After acetonitrile protein precipitation, DMB and dimethylvaleric acid (DMV) internal standard were derivatized to benzylamides, chromatographed on a Hydro-RP column with acetonitrile, water, and 0.1% formic acid, and detected by electrospray positive-mode ionization mass spectrometry. The assay was accurate (97-107%) and precise (3.4-6.2%) between 100 and 10,000ng/mL. Recovery from plasma was >62%. Plasma freeze-thaw and room temperature stability were acceptable.     

Metachromatic assay for the quantitative determination of bacterial endotoxins

A metachromatic dye, 1,9-dimethylmethylene blue (DMB) reacts with bacterial endotoxins. This interaction results in a shift of the absorption maximum of DMB to shorter wavelengths. The findings indicate that the negatively charged lipid moiety of the endotoxic lipopolysaccharide reacts with DMB. The lowest amount of endotoxin detectable by the procedure described here is approximately one microgram. The dye could not be used in the presence of serum components. DMB mixed to column chromatographic effluent showed good resolution in continuous monitoring of endotoxin components leaving the column.     

The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole.

The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.     

Electron Transport and Nonlinear Optical Properties of Substituted Aryldimesityl Boranes: A DFT Study

A comprehensive theoretical study was carried out on a series of aryldimesityl borane (DMB) derivatives using Density Functional theory. Optimized geometries and electronic parameters like electron affinity, reorganization energy, frontiers molecular contours, polarizability and hyperpolarizability have been calculated by employing B3PW91/6-311++G (d, p) level of theory. Our results show that the Hammett function and geometrical parameters correlates well with the reorganization energies and hyperpolarizability for the series of DMB derivatives studied in this work. The orbital energy study reveals that the electron releasing substituents increase the LUMO energies and electron withdrawing substituents decrease the LUMO energies, reflecting the electron transport character of aryldimesityl borane derivatives. From frontier molecular orbitals diagram it is evident that mesityl rings act as the donor, while the phenylene and Boron atom appear as acceptors in these systems. The calculated hyperpolarizability of secondary amine derivative of DMB is 40 times higher than DMB (1). The electronic excitation contributions to the hyperpolarizability studied by using TDDFT calculation shows that hyperpolarizability correlates well with dipole moment in ground and excited state and excitation energy in terms of the two-level model. Thus the results of these calculations can be helpful in designing the DMB derivatives for efficient electron transport and nonlinear optical material by appropriate substitution with electron releasing or withdrawing substituents on phenyl ring of DMB system.