用氨基葡萄糖含量的测定值间接表示红曲菌固态发酵过程中生物量的研究

【目的】为准确快速地了解紫色红曲菌固态发酵中生物量的变化,【方法】采用理化方法测定菌体量和氨基葡萄糖含量,研究了不同培养时间、培养基组成、培养方式下菌体量与氨基葡萄糖含量的关系,建立生物量和氨基葡萄糖含量的换算关系式;构建关联该菌固态培养物近红外光谱数据与实测氨基葡萄糖含量的PLS模型。【结果】建立了可通过近红外光谱法测定氨基葡萄糖来快速预测固态发酵生物量的方法,其中最优近红外模型的校正集内部交叉验证均方根误差(RMSECV)为0.209 4,预测集相关系数(Rp)和均方根误差(RMSEP)分别为0.993 4和0.217 3;同时利用所建的换算关系式也大大提高了生物量计算的准确性。【结论】基于所建立的生物量和氨基葡萄糖的换算关系式,利用近红外光谱法可以快速并且较准确地测定紫色红曲菌固态发酵过程中生物量的变化。     

Glucosamine measurement as indirect method for biomass estimation of Cunninghamella elegans grown in solid state cultivation conditions

Glucosamine measurement has been tested as the indirect method to estimate the biomass produced by Cunninghamella elegans during solid state cultivation (SSC). The independence of this cell constituent content from the age and the conditions of the culture have been verified. The influence of the medium composition, in particular the nature of the carbon source on glucosamine amount is presented. Glucosamine can be considered as a well-adapted biomass indicator, with the necessity to establish for each medium tested a prior correlation between biomass and glucosamine amount. This correlation should be defined in submerged conditions before applying the biomass estimating method in SSC.     

Composition of Pseudomonas aeruginosa slime

1. The slime produced by eight strains of Pseudomonas aeruginosa on a number of different media was demonstrated to be qualitatively the same. Small quantitative differences may be occasioned by differences in the extraction procedure, the growth medium or the strain of organism used. 2. The slime was shown to be predominantly polysaccharide with some nucleic acid material and a small amount of protein. 3. The hydrolysed polysaccharide fraction consists mainly of glucose with smaller amounts of mannose. This accounts for some 50-60% of the total slime. In addition, there is some 5% of hyaluronic acid. The nucleic acid material represents approx. 20% of the total weight, and is composed of both RNA and DNA. 4. Minor components are protein, rhamnose and glucosamine, the protein being less than 5% of the total. 5. Hyaluronic acid is produced in greater quantities from nutrient broth than from chemically defined media, and is more firmly attached to the cells than the other components.     

Reliability, repeatability and accuracy of the falling head method for hydraulic conductivity measurements under laboratory conditions

The aim of this study was to verify under lab conditions the reliability, repeatability and accuracy of the falling head method (FHM) for hydraulic conductivity measurements. The FHM is a reliable procedure that has slight variations (less than 10%) in repeated measurements and turns out to be a reliable technique to record the hydraulic conductivities typically described for clogged and unclogged subsurface-flow constructed wetlands (from 4 to ca. 360 m/day). The accuracy of the method is acceptable considering difficulties in the measurement of hydraulic conductivity in highly conductive media. Accordingly, results show measurement deviations of 20% when compared with a laboratory constant head method for highly conductive media (higher than 250 m/day), and 80% for media with low hydraulic conductivity (lower than 50 m/day). The main conclusion of the present paper is that of the FHM is a reliable and repeatable technique for hydraulic conductivity measurements and it is accurate enough for on-site clogging assessment in full-scale constructed wetlands.     

东北黑土氨基糖的矿化动态及其对外源物质添加的响应

采用间歇淋洗好气培养法研究了东北黑土中3种不同微生物来源氨基糖(氨基葡萄糖、胞壁酸和氨基半乳糖)的矿化动态以及对葡萄糖添加和葡萄糖与氮肥配施的响应.结果表明:土壤中不同种类的氨基糖具有不同的矿化特征.培养期间胞壁酸含量减少25.4%而氨基葡萄糖含量降低7.1%,表明细菌来源的胞壁酸在土壤中的矿化速率快于真菌来源的氨基葡萄糖,但氨基葡萄糖的矿化数量(68.4 mg·kg^-1)显著高于胞壁酸(15.4 mg·kg^-1).葡萄糖添加以及葡萄糖与氮肥配施均显著提高了土壤中氨基葡萄糖和胞壁酸的含量,但两种处理的影响有所不同.相比之下,氨基半乳糖在土壤中矿化较慢,并且受外源物质的影响较小,表现出较高的稳定性.     

An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5'-UMP and glucosamine, in which yeast cells catalyze the conversion of 5'-UMP to 5'-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5'-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5'-UMP and 100 mM GlcNAc.     

Estimation of mycelial growth of basidiomycetes by means of chitin determination

After hydrolysis of chitin in 6 M HCl, the glucosamine produced was assayed colorimetrically. The pH of the hydrolysate was adjusted to a value close to three by addition of Na acetate; this procedure avoids the elimination of excess acid by evaporation under reduced pressure or freeze-drying. Under these conditions, the amount of glucosamine determined by the assay represented an average of 90% of the amount which would result from a total hydrolysis of the chitin. The method was used to assay the chitin in the mycelia of basidomycetes obtained in vitro. The measured amount of glucosamine was proportional to the mycelial biomass and allowed the estimation of fungal growth.     

MALDI-TOF MS技术在侵袭性丝状真菌快速鉴定中的应用

探索不同培养基、不同培养时间和不同蛋白质提取方法对侵袭性丝状真菌质谱鉴定准确率的影响, 旨在提高基质辅助激光解析电离飞行时间质谱技术鉴定侵袭性丝状真菌的准确率。

采用分子生物学方法为金标准, 同时运用基质辅助激光解析电离飞行时间质谱技术对所收集临床丝状真菌进行鉴定。根据分子生物学的鉴定结果, 去除VITEK-MS v3.0数据库中没有的菌株, 其余菌株接种在沙氏葡萄糖琼脂(SDA)、马铃薯葡萄糖琼脂(PDA)和察氏培养基(CA)3种不同的培养基中, 采用2种不同的蛋白质提取方法(甲酸乙腈法和磁珠法), 获得了不同培养时间点(2、3、5、7和9 d)的特异性质谱指纹图谱。

不同丝状真菌蛋白质提取方法进行比较, 甲酸乙腈法总鉴定准确率为79.8%, 磁珠法总鉴定准确率为77.5%, 2种丝状真菌蛋白质提取方法的质谱鉴定准确率差异无统计学意义(χ²=1.040, P=0.308)。不同培养基进行比较, SDA培养基总鉴定准确率为90.7%, PDA培养基总鉴定准确率为81.4%, CA培养基总鉴定准确率为67.4%, 3种不同培养基的丝状真菌质谱鉴定准确率差异有统计学意义(χ²=36.609, P < 0.001), 其中使用SDA培养基鉴定准确率最高, 使用CA培养基鉴定准确率最低(SDA vs PDA, χ²=7.748, P=0.005;SDA vs CA, χ²=35.131, P < 0.001;PDA vs CA, χ²=10.994, P=0.001)。不同培养时间进行比较, 丝状真菌培养2、3、5、7和9 d后的质谱总鉴定准确率分别为64.3%、88.4%、89.1%、79.1%和78.3%, 不同培养时间的质谱鉴定准确率差异有统计学意义(χ²=32.274, P < 0.001)。培养3 d和培养5 d的质谱鉴定准确率优于培养7 d和培养9 d的质谱鉴定准确率, 培养2 d的质谱鉴定准确率明显低于其他时间(3 d vs 5 d, χ²=0.039, P=0.844;7 d vs 9 d, χ²=0.023, P=0.879;3 d vs 7 d, χ²=4.095, P=0.043;2 d vs 9 d, χ²=6.139, P=0.013)。

侵袭性丝状真菌使用SDA培养基培养3 d, 运用甲酸乙腈法提取蛋白质进行质谱鉴定最理想。

     

Improved and simplified liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized glucosamine in human plasma

Glucosamine is an amino monosaccharide reagent. It is difficult to assay using typical reversed-phase column due to the early elution, by optimizing the chromatographic conditions, especially the analytical column and the mobile phase composition, an improved analytical method was developed and validated, which offers rapid, sensitive and specific determination of glucosamine in human plasma. Following protein precipitation, the analyte and internal standard (valibose) were separated using an isocratic mobile phase on an Inertsil CN-3 column and detected by mass spectrometry in the multiple reaction monitoring mode using the respective precursor to product ion combinations of m/z 180/72 for glucosamine and m/z 252/198 for valibose. The chromatographic time was just 4.2 min for each sample, which made it possible to analyze more than 120 human plasma samples per day. The method exhibited a linear dynamic range of 4.00-4000 ng/mL for glucosamine in human plasma. The lower limit of quantification (LLOQ) was 4.00 ng/mL with a relative standard deviation of less than 10.9%. Acceptable precision and accuracy were obtained for the plasma concentrations over the standard curve range. By monitoring the two different MRM transitions, it was proved that no endogenous glucosamine was found in human plasma. The validated method has been successfully used to analyze human plasma samples for application in a bioequivalence study.     

Isolation and characterization of a glucosamine-requiring mutant of Escherichia coli K-12 defective in glucosamine-6-phosphate synthetase

A mutant was isolated from Escherichia coli K-12 which requires glucosamine or N-acetylglucosamine for growth. Depriving the mutant of glucosamine resulted in a rapid loss of viability of the cells, followed by a decrease in the turbidity of the culture. When the mutant cells were resuspended in broth media containing 10% sucrose, the rod-shaped cells became spheroplasts. However, the presence of sucrose in the media did not prevent the cells from losing their viability. This mutant was shown to be deficient in the activity of l-glutamine:d-fructose-6-phosphate aminotransferase (EC 2.6.1.16). The activity of the deaminating enzyme, 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (EC 5.3.1.10), appeared to be normal in this mutant. The position of the mutation has been determined to be at the 74th min of the Taylor and Trotter map, as shown by cotransduction with phoS (90%) and ilv (25%) by using bacteriophage P1.     

Chitooligosaccharides antagonize the cytotoxic effect of glucosamine

Chitooligosaccharides (COS) are partially hydrolyzed compounds derived from chitosan that exhibit a number of biological activities, including antitumor, antibacterial and antifungal properties. In this work, we examined the cytotoxicity of pure COS and oligomers A, B and C (solutions composed of different amounts of COS) produced by enzymatic hydrolysis using a crude enzyme extract produced by the fungus Metarhrizium anisopliae. The antiproliferative effect of these molecules was analyzed using tumor cell lines (HepG2 and HeLa cells) and in a normal cell line (3T3). The antioxidant activity was analyzed in several in vitro experiments. Glucosamine showed higher toxicity (approximately 92%) to all cell lines studied. However, the oligomers obtained after hydrolysis demonstrated no toxic effects on the normal cells (3T3). Furthermore, we showed that a small amount of other COS can decrease the cytotoxic effect of glucosamine against 3T3 cells, indicating that glucosamine could be used as an antitumor drug in the presence of other COS. In addition, different effects were found in antiproliferative assays, which depended on the COS composition in the oligomers (A, B and C), showing that a combination of them may be essential for developing antineoplastic drugs. Superoxide anion scavenging was the main antioxidant activity demonstrated by the COS and oligomers. This activity was also dependent on the oligomer composition of the chitosan hydrolysates. Further work will identify the ideal proportions of COS and glucosamine for maximizing the effects of these biological activities.     

Determination of glucosamine in impure chitin samples by high-performance liquid chromatography

A simple, rapid, selective, and specific high-performance liquid chromatography (HPLC) method was developed to quantitate glucosamine, and its application for estimating purity of chitin was investigated. The chromatographic separation was achieved using a reversed-phase C8 column, pre-column derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and ultraviolet detection (lambda=254 nm). The mobile phase consisted of CH3CN and H2O. The optimum conditions of acid hydrolysis of chitin (concentration of HCl, temperature, and heating time) was obtained by performing the orthogonal array design (OAD) procedure and the released glucosamine was determined by the above HPLC method. The accuracy of the method was checked by the standard addition technique. The method was found to be specific with good linearity, accuracy, precision, and well suited for quantitation of glucosamine and determination of the purity of chitin in biological materials and food products.     

Glucosamine and glucose induce insulin resistance by different mechanisms in rat skeletal muscle

It has been hypothesized that glucose-induced insulin resistance is mediated by accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs). In a previous study on rat epitrochlearis muscles incubated with high concentrations of glucose and insulin (Kawanaka K, D-H Han, J Gao, LA Nolte, and JO Holloszy. J Biol Chem 276: 20101-20107, 2001), we found that insulin resistance developed even when the increase in UDP-Hex-NAcs was prevented. Furthermore, actinomycin D completely prevented glucose-induced insulin resistance despite a greater accumulation of UDP-HexNAcs. In the present study, we used the same epitrochlearis muscle preparation, as well as the rat hemidiaphragm, to determine whether, like glucose, glucosamine causes insulin resistance by an actinomycin D-inhibitable process. Incubation of diaphragm muscles with 10 mM glucosamine for 3 h resulted in an approximately fivefold increase in UDP-HexNAcs, an approximately 50% reduction in insulin responsiveness of glucose transport, and a 58% reduction in ATP concentration. These effects of glucosamine were not prevented by actinomycin D. Incubation of epitrochlearis muscles with 20 mM glucosamine for 3 h or with 10 mM glucosamine for 5 h also caused large decreases in insulin responsiveness of glucose transport but with no reduction in ATP concentration. Actinomycin D did not prevent the glucosamine-induced insulin resistance. The insulin-induced increases in tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the binding of PI 3-kinase to IRS-1 were decreased approximately 60% in epitrochlearis muscles exposed to glucosamine. This is in contrast to glucose-induced insulin resistance, which was not associated with impaired insulin signaling. These results provide evidence that glucosamine and glucose induce insulin resistance by different mechanisms.     

Qualitative and quantitative analysis of reducing carbohydrates by radiochromatography on ion-exchange papers

A highly specific method for qualitative and quantitative analysis of reducing carbohydrates has been described. The method involves the measurement of the amount of tritium found in [³H]sodium borohydridereduced sugars following their paper chromatographic separation on ion-exchange papers. These procedures may be used to quantitate all of those components in 5 μl of a mixture which are present in amounts ranging from 0.1 to 10,000 nmoles.The quantitation gives approximately 10% accuracy at the lower end of the sensitivity range.     

The acid mucopolysaccharides of cattle retina

1. Two polysaccharides were isolated from the interstitial matrix surrounding the photoreceptor cells of cattle retina. They were liberated from this region of the tissue in a soluble form after agitation of whole retinas in 0.9% sodium chloride. One, which comprises two-thirds of the polysaccharides present, is a hyaluronidase-sensitive ;half-sulphated' chondroitin sulphate containing uronic acid, galactosamine and sulphate in the molar proportions 1.27:1.0:0.54. The other is a hyaluronidase-resistant non-sulphated heteropolysaccharide for which the name sialoglycan is proposed. It contains galactose, glucosamine and sialic acid in the molar proportions 2.4:1.0:0.4. Both polysaccharides contain only small amounts of nitrogen in excess of the amount calculated from their amino sugar and sialic acid content. 2. A similar combination of mucopolysaccharides is associated with the pigment epithelial-cell layer but in quantities only one-fifth of those present in the adjacent matrix area. 3. The ease with which they are released into aqueous media is consistent with the assumption that they are present in the extracellular spaces in both of these tissue layers. 4. The retinal residue left after removal of the two soluble polysaccharides is rich in amino sugar- and sialic acid-containing polymers, which appear to be firmly bound to the tissue fragments. 5. About one-third of the sialic acid and one-tenth of the amino sugar could be extracted with chloroform-methanol. The components in this fraction were tentatively identified as gangliosides. 6. Digestion of the chloroform-methanol-insoluble residue with Pronase yielded as the principal product a heteropolysaccharide containing 16.5% of glucosamine, 24.3% of neutral sugar (galactose plus fucose) and 18.1% of sialic acid. This substance has been classified as a sialoglycan of composition similar to (but not identical with) that of the soluble one isolated from the matrix area of the tissue.     

Automatic Detection of Calcaneal-Fifth Metatarsal Angle Using Radiograph: A Computer-Aided Diagnosis of Flat Foot for Military New Recruits in Taiwan

Flatfoot (pes planus) is one of the most important physical examination items for military new recruits in Taiwan. Currently, the diagnosis of flatfoot is mainly based on radiographic examination of the calcaneal-fifth metatarsal (CA–MT5) angle, also known as the arch angle. However, manual measurement of the arch angle is time-consuming and often inconsistent between different examiners. In this study, seventy male military new recruits were studied. Lateral radiographic images of their right and left feet were obtained, and mutual information (MI) registration was used to automatically calculate the arch angle. Images of two critical bones, the calcaneus and the fifth metatarsal bone, were isolated from the lateral radiographs to form reference images, and were then compared with template images to calculate the arch angle. The result of this computer-calculated arch angle was compared with manual measurement results from two radiologists, which showed that our automatic arch angle measurement method had a high consistency. In addition, this method had a high accuracy of 97% and 96% as compared with the measurements of radiologists A and B, respectively. The findings indicated that our MI registration measurement method cannot only accurately measure the CA–MT5 angle, but also saves time and reduces human error. This method can increase the consistency of arch angle measurement and has potential clinical application for the diagnosis of flatfoot.     

Determination of glucosamine sulfate in human plasma by precolumn derivatization using high performance liquid chromatography with fluorescence detection: its application to a bioequivalence study

A simple, rapid, selective and specific high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of glucosamine sulfate in human plasma and application to a bioequivalence in healthy volunteers. Precipitation of plasma was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. After vortex mixing and centrifugation, the supernatant was transferred and derivatized with 9-fluorenylmethoxycarbonyl chloride-acetonitrile solution in borate buffer (pH=8.0) at 30 degrees C for 30 min. The chromatographic separation was performed on a Diamonsil C18 column (150.0 mmx4.6 mm, 5 microm) with a mobile phase gradient consisting of water and acetonitrile at a flow rate of 1 mL/min. The method was linear in the range of 0.1-10.0 microg/mL with a correlation coefficient (r) of 0.9996. The limit of detection was 15 ng/mL. Inter- and intra-day precisions were 相似文献   

Development and validation of a sensitive HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma

A sensitive and specific HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma has been developed and validated. Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid using a polymer-based amino high-performance liquid chromatography (HPLC) column and a water/acetonitrile mobile phase elution gradient, with d-[1-(13)C]glucosamine as the internal standard. Detection was performed by mass spectrometry, using an electrospray source and employing multiple reaction monitoring to separately monitor glucosamine and the internal standard. The limit of quantification of the method was 10ng/ml of glucosamine and the calibration curve showed a good linearity up to 1000ng/ml. The precision (R.S.D.) and the accuracy (bias) of the method at the limit of quantification were 13.8 and 4.0%, respectively, and the mean recovery of glucosamine at three concentration levels was 101.6+/-5.7%. The method was applied for the determination of glucosamine concentrations in human plasma samples collected from untreated healthy volunteers and, in a separate bioavailability study, to evaluate plasma glucosamine pharmacokinetics profiles after oral administration of crystalline glucosamine sulfate.     

Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells

The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal alpha1-6-linked fucose; 37% of the antenna were found to be substituted with terminal alpha2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations.     

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.