Histochemical demonstration of two subtypes of O-linked oligosaccharides in the rat gastric glands

The gastric glands synthesize glycoproteins whose oligosaccharides are linked to the peptide core mainly by the O-glycosidic bond, specifically removed by beta-elimination procedure. Our aim was to research the possibility of the existence of two subtypes of O-linked oligosaccharides with a different behavior to the removal procedure. The lectins from peanut (PNA) and Maackia amurensis (MAA-I) were histochemically used as markers of the O-linked oligosaccharides. Sections were also pretreated with beta-elimination and/or peptide N-Glycosidase F (PNGase-F) for the specific removal of O- and N-linked oligosaccharides, respectively. The lectin GNA, which mainly labels to N-linked oligosaccharides, was used to test the correct working of PNGase-F. To test the possibility that the beta-elimination treatment could remove the terminal sialic acid residues, the lectin LFA was used. The surface epithelium was negative to PNA, while it became strongly positive when beta-elimination was performed for 1 day. This staining was resistant to PNGase-F, suggesting that PNA was labeling to O-linked oligosaccharides. However, after beta-elimination for 5 days this staining is not observed. A similar pattern appeared with MAA-I. We propose the existence of two subtypes of O-linked oligosaccharides: labile and resistant. The labile O-linked oligosaccharides are removed with beta-elimination for 1 day, unmasking the PNA-positive oligosaccharides. These oligosaccharides are resistant O-linked oligosaccharides because staining is abolished with longer treatment of beta-elimination. The results with MAA-I also support this suggestion. In summary, the labile O-linked oligosaccharides are removed with short treatment, while the resistant O-linked oligosaccharides need a stronger procedure (for 5 days).     

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic‐Acid Schiff, Alcian Blue pH 2.5, High‐Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA‐I, WGA, SBA, BSI‐B4, ConA, AAA, UEA‐I, LTA, LFA, MAA‐II, SNA) combined with chemical and enzymatic pre‐treatments (β‐elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O‐linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin‐binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co‐distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.     

Specific techniques for the identification of O-acylated sialic acids in colonic mucins

The development of methodology for the histochemistry of mucins based upon their PAS reactivity is discussed in terms of mechanism, specificity and application. Two new histochemical methods (PB/KOH/PAS and PAT/KOH/PAS), supplemented by a variety of new and standard histochemical techniques, and correlated by parallel chemical studies, were used to demonstrate and identify C₄ and side chain O-acylated sialic acids in colonic epithelial mucins. The application of these methods in the field of histopathology is discussed.     

Differentiation of isomeric amino acid residues in proteins and peptides using mass spectrometry

Characterization and differentiation of isomers in biological macromolecules using mass spectrometry is one of the most significant challenges facing scientists in the field. The capability of high‐resolution MS instruments along with the development of new fragmentation methods now provides the ability to indirectly differentiate between some isomers. This ability has enabled mass spectrometry to evolve into a multidisciplinary technique incorporating areas such as pharmaceutical research, proteomics, polymer science, medicine, environmental chemistry, and recently archeology. This article aims to review recent developments in mass spectrometry methodologies in the identification of structural and spatial isomers in biological macromolecules, such as aspartic acid and isoaspartic acid (Asp/IsoAsp), leucine and isoleucine (Leu/Ile), glutamic acid and γ‐glutamic acid, and D/L enantiomers. © 2012 Wiley Periodicals, Inc. Mass Spec Rev 31:609–625, 2012     

高效液相色谱法测定复方苯甲酸软膏中苯甲酸及水杨酸的含量

研究用高效液相色谱法测定复方苯甲酸软膏的含量。用甲醇做溶剂,70℃水浴溶解软膏,冰浴析出基质,使基质与水杨酸、苯甲酸分离,采用Zorbax-SB C_(18)柱,0.1Mol/L磷酸氢二钠:甲醇(60:40)作为流动相,流速0.8mL/min。在240nm波长处,用外标法测定含量。苯甲酸、水杨酸浓度分别在0.01～0.2mg/mL范围内线性关系良好,R=0.9996。最低检测限为200ng/mL。日内精密度为0.01%～0.04%,0.019%～0.11%,日间精密度为0.045%～0.74%,0.039%～0.19%。方法回收率苯甲酸为:98.31% (n=4),RSD0.79%。水杨酸为:98.54% (n=4),RSD为1.62%。基质凡士林对苯甲酸、水杨酸的测定无干扰。试验证明本方法操作简便、快速,结果准确,可用于该制剂的含量测定。     

气相色谱法测定食品中山梨酸、苯甲酸的改进

目的建立食品中山梨酸、苯甲酸检测方法并对其前处理进行合理的改良。方法样品经盐酸酸化,用乙醚提取,接着用氯化钠酸性溶液洗涤2次后将提取液转入无水硫酸钠溶液中蒸发近干,残渣用丙酮溶解;用色谱柱分离,具氢火焰离子化检测器进行检测。结果本法山梨酸和苯甲酸相对偏差分别为3.71%和2.11%;加标回收率分别为87.0%-95.2%和97.0%-103.1%,远远高于国标法中的回收率。结论本方法样品前处理操作简单方便,准确度和灵敏度高,适用于食品中山梨酸、苯甲酸的测定。     

A SEM study of the antenna and mouthparts of Omosita colon (Linnaeus) (Coleoptera: Nitidulidae)

There are direct relationships between the behavioral mechanisms and sensilla. To obtain a better understanding of the behavioral mechanisms in Omosita colon (Linnaeus) (Coleoptera: Nitidulidae), we investigated the types, quantities, and distribution of sensilla on the antenna and mouthparts of O. colon by scanning electron microscopy. The clavate antenna comprised the scape, pedicel, and nine segment flagellomeres and had six types of sensilla, including two subtypes of sensilla chaetica (SC), three subtypes of sensilla basiconica (SB) and sensilla trichodea (ST), and one type of sensilla cavity, sensilla styloconica, and Böhm bristles (BB). The chewing mouthparts of O. colon consist of the labrum, mandible, maxillae, labium, and hypopharynx and had seven types sensilla, including two subtypes of SC and sensilla placodea, seven subtypes of SB, and one type of BB, ST, sensilla coeloconica, and sensilla campaniformia. In this research, we also deduced the relationships between the sensilla on the antenna and mouthparts and their functions.     

工作场所空气中磷酸测定方法的研究

建立了用抗坏血酸为还原剂的分光光度法测定工作场所空气中磷酸的方法。微孔滤膜采集空气中的磷酸雾,水洗脱后,在酸性溶液中,与钼酸铵生成磷钼杂多酸,以抗坏血酸还原,生成磷钼蓝,在700nm波长下用分光光度计测定。方法曲线的相关系数r=0.9999,线性范围0～200μg,检出限0.068μg/mL,相对标准偏差为0.17%～0.96%,加标回收率为94.0%～103.9%。用本方法测定工作场所空气中磷酸,效果很好、线性关系良好、检出限低、准确度及精密度高。     

Comparative histochemical analysis of glycoconjugates in the nasal vestibule of camel and sheep

While Corriedale sheep survive in a wide range of climates, which prevents them to specialize for one climatic condition only, dromedary camels strictly adapted to desert areas. This demands more adaptive mechanisms to hot, dry conditions in camels than in sheep. Being the entrance of the nasal cavity, nasal vestibule is subjected to various environmental stressors. A protective way is the lining epithelium which is cornified in camel, but not in sheep. Mucus nasal secretions also play a key role in the protection of underlyings. Additionally, arterio‐venous anastomosis is present in the lamina propria of the nasal vestibule of camel. In the present paper, sugar residues in the nasal vestibule of camel were analyzed and compared with those of sheep using 14 types of lectins to explore the distribution of glycoconjugates that may help the function of camel nasal vestibule in desert environment. In camel, none of the lectins could label the basal cells of the vestibular epithelium, although the basal cells reacted with six lectins in sheep. In camel, LEL and RCA‐120 markedly labeled the luminal surface. WGA, DBA, SBA, and VVA produced marked intensities on the luminal surface in sheep. The mucous glands reacted with six lectins: WGA, s‐WGA, VVA, PNA, PHA‐E, and PHA‐L in camel, while all lectins used except s‐WGA and PHA‐E reacted in the sheep. In summary, great differences are observed in the glycoconjugate expression between camel and sheep. This suggests that these glycoconjugate are related to camel's tolerance for environmental stressors.     

Development of the vomeronasal receptor epithelium and the accessory olfactory bulb in sheep

The morphological development of the vomeronasal organ (VNO) and accessory olfactory bulb (AOB) of the sheep from anlage to birth were studied by classical and histochemical methods using embryos and fetuses obtained from an abattoir with ages estimated from crown-to-rump length. Both VNO and AOB developed in a biologically logical sequence and completed their morphological development around day 98, at entry into the last third of the gestation period. A lectin with specificity for oligomeric N-acetylglucosamine labeled the sensory epithelium of the VNO, the vomeronasal nerves, and the nervous and glomerular layers of the AOB before birth. These results suggest that the vomeronasal system, which is well developed and functional in adult sheep, may be able to function at or even before birth in these animals (whereas in rodents, for example, this is precluded by the AOB not completing its development until after birth).     

Investigation of polyelectrolyte for electrochemical detection of uric acid in presence of ascorbic acid

Uric acid (UA) was detected in the presence of ascorbic acid (AA) at GC electrode by cyclic voltametry (CV) and differential pulse voltametry (DPV) in aqueous media of cationic polyelectrolyte (poly(diallyldimethylammonium chloride) (PDDA)). Both, UA and AA are anionic nature and electro-static attraction with cationic solution. This lowered their oxidation potentials and increased anodic current. In CV studies, the UA oxidation potential was decreased by 400 mV in the presence of PDDA along with increase in peak current. Effect of PDDA and pH on E_pa and I_pa were also studied. About 360 mV difference in oxidation peak potentials was observed for AA and UA in PDDA media, which established a quick method for their simultaneous determination. The detection limit of UA in the presence of 200 folds AA was found as 1 μM with correlation coefficient of 0.994 and sensitivity of 0.05 μA μM⁻¹. The proposed method has been also applied for determining the UA in human urine without any pretreatment, and found to be satisfactory.     

不同花色映山红花中熊果酸和齐墩果酸的含量比较

结合超声技术提取不同花色映山红花中熊果酸和齐墩果酸,建立RP—HPLC法测定其含量。采用KromasilC18色谱柱（250mm×4．6mm,5μm）,流动相为甲醇-0．2％磷酸水溶液（88：12）,光电二极管阵列检测器,检测波长210nm,流速0．8mL／min,柱温30℃。以保留时间和紫外光谱对分离出的组分予以定性确证,用峰面积进行定量。组分的质量浓度与其峰面积在一定的范围内呈现良好的线性关系,相关系数均为0．9999,熊果酸进样量在0．443～7．088μg,齐墩果酸进样量在0．247～3．952μg时呈现良好的线性关系,平均加样回收率分别为98．53％和98．36％,RSD分别为1．3％和1．5％（n=5）。方法灵敏、准确,重现性好,可用于不同花色映山红花中熊果酸和齐墩果酸含量的测定。     

Method for localization of sialic acid on cell surface and cell interior in peripheral blood: a pilot study

This article presents a method for identification and localization of cell surface and intracellular sialoglycoconjugates of peripheral blood cells. To reveal cell surface conjugates, a sample of peripheral blood was incubated with lectin after centrifugation and rinsing. For intracellular localization in leukocytes, RBCs were lysed and the membranes were permeabilized prior to cytochemical reaction. Fluorescein isothiocyanate conjugated lectins were used for visualization in fluorescence microscope. All lectins bound specifically to the surface of erythrocytes. Confocal microscopy showed surface and intracellular labeling of permeabilized leukocytes. A part of the signal in eosinophils originated from binding of anionic fluorophore to cationic granular proteins.     

Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

Background: Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period. Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells (hiPS-CM), researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM. In our previous studies, we separately supplemented two polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) or arachidonic acid (AA), to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation, fatty acid metabolism, and cellular adhesion. The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force. Methods: This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM. In addition to the contractile force and mRNA measurements used in the previous study, we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis, measured glucose and fatty acids metabolites, and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation. Results: It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors, and the supplementations may activate the CD36_­ERRγ metabolic pathway. DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters. The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant. AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of P300 and CD36. However, finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings. Conclusion: DHA and AA, as having been supplemented in infant formulas, have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually, although they were able to enter the cellular metabolic system. The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM, which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.     

Development and characterization of a new nickel(II) ion selective optode based on 2-amino-1-cyclopentene-dithiocarboxylic acid

A highly sensitive and selective optical membrane sensor was prepared for the determination of ultra trace amount of Ni²⁺ ions. The plasticized PVC membrane incorporating dibuthylphthalate and 2-amino-1-cyclopentene-dithiocarboxylic acid (ACDA) as a chromoionophore works on the basis of a cation-exchange mechanism and shows a significant absorbance signal change on exposure to acidic solution containing nickel(II) ion. The sensor displays a calibration response for Ni²⁺ ion over a wide concentration range of 3.1 × 10⁻⁸-8.0 × 10⁻³ M, and a response time of 3 min. In addition to high stability, reproducibility and relatively long working lifetime, the sensor possesses good selectivity for nickel(II) ion over several common diverse ions. The sensor was successfully applied to determine the traces of Ni²⁺ ion in some water samples.     

高效液相色谱法测定复方花针颗粒中绿原酸的含量

报道了采用HPLC法测定复方花针颗粒中绿原酸的含量。色谱柱为ZOR-BAXSB-C18分析柱,流动相为乙腈-2％冰醋酸(6.5:93.5),检测波长为 326nm。实验结果表明,该方法简便、准确,灵敏度高,重现性好。     

游泳池水中氰尿酸快速检测仪性能评价

对游泳池水中氰尿酸快速检测仪的反应时间、线性、试剂批次、不同操作人员对检测结果的影响,以及仪器波长示值误差和重复性、仪器示值误差和重复性以及样品加标回收率等性能指标进行了系统的考察。实验结果表明:氰尿酸在20.0～100.0 mg/L检测范围内时,检测仪标准曲线的线性相关系数r大于0.9998;不同批次试剂检测相对误差小于±5%;不同操作人员检测同一样品的相对误差小于±10%;3台检测仪波长示值误差范围为-0.05 nm～0.53 nm,波长重复性范围为0.02 nm～0.05 nm; 3台仪器设备对20.00 mg/L和50.00 mg/L两浓度标准溶液测定的示值误差均小于±5%,重复性均小于5%;水样的低、中、高浓度加标回收率范围为86.3.0%～111.6%。     

微柱液相色谱法同时检测食品中苯甲酸和山梨酸

通过自构建的微柱液相色谱系统上色谱条件的优化,建立了一种同时检测食品中苯甲酸和山梨酸的新方法。食品中苯甲酸和山梨酸经提取后,6分钟内达到基线分离,与常规高效液相色谱法相比,分析时间缩短,流动相消耗减少。在1～25 mg/L浓度范围内线性关系良好,相关系数分别为0.9999和0.9997。苯甲酸和山梨酸的回收率均在94.7%～103.1%之间,三次平行加标回收率测试,相对标准偏差分别为1.12%和1.29%。该方法稳定性好,回收率高,能快速、准确的检测食品中苯甲酸和山梨酸。     

Effects of the ascorbic acid supplementation on NADH-diaphorase myenteric neurons in the duodenum of diabetic rats.

We assessed the ascorbic acid (AA) supplementation on the myenteric neurons in the duodenum of rats. Fifteen rats with 90 days of age were divided into three groups: control (C), diabetics (D) and ascorbic acid treated diabetics (DA). After 120 days of daily treatment with AA, the duodenum was submitted to the NADH-diaphorase (NADH-d) histochemical technique, which allowed us to evaluate the neuronal density in an area of 8.96 mm2 for each duodenum, and also to measure the cellular profile area of 500 neurons per group. The supplementation promoted an increase on AA levels. The neuronal density (p < 0.05) was higher in the group DA when compared to group D. There were no significant differences in the neuronal areas, when we compared groups C (204 +/- 16.5) and D (146.3 +/- 35.84) to groups D and DA (184.5 +/- 5.6) (p > 0.05). The AA-supplementation avoided the density reduction of the NADHd myenteric neurons in the duodenum of diabetic rats.