Glycan modifying enzymes in luminal fluid of rat epididymis: are they involved in altering sperm surface glycoproteins during maturation?

Testicular spermatozoa undergo morphological and biochemical alterations, collectively termed epididymal maturation, in the intraluminal environment of epididymis. As a result of these modifications, the spermatozoon becomes a motile and functionally competent cell capable of undergoing capacitation and binding to the zona pellucida, the extracellular coat that surrounds the mammalian oocyte. Although details of all the changes are not fully known, several studies provide evidence suggesting that sperm plasma membrane undergoes extensive biochemical changes, including organization and modification of surface glycoproteins as spermatozoa transit from the proximal to the distal epididymis. In this article, I have attempted to summarize results with two sets of glycoprotein (glycan)-modifying enzymes, namely, glycohydrolases (hydrolytic enzymes) and glycosyltransferases (synthetic enzymes) present in the epididymal luminal fluid (LF). The in vitro experimental approaches described in this report demonstrate that: 1) a PNA-positive glycoprotein(s) (containing O-linked glycan) of 135-150 kDa subunit molecular mass which is present on the surface of caput (but not the cauda) spermatozoa can be degalactosylated by the enzymatic digestion with LF beta-D-galactosidase; and 2) an N-linked glycan chain(s) which is present on a sperm surface glycoprotein (apparent subunit molecular mass of 86 kDa) can be fucosylated in vitro when distal caput sperm (or sperm plasma membrane-rich fractions) are incubated in the presence of a nucleotide sugar (GDP[(14)C]fucose). Combined, these results strongly suggest a role for the glycan-modifying enzymes in degalactosylation and fucosylation of sperm surface glycoproteins during epididymal transit.     

Traffic through the golgi apparatus as studied by radioautography

The ability to radiolabel biological molecules, in conjunction with radioautographic or cell fractionation techniques, has brought about a revolution in our knowledge of dynamic cellular processes. This has been particularly true since the 1940's, when isotopes such as ³⁵S and ¹⁴C became available, since these isotopes could be incorporated into a great variety of biologically important compounds. The first dynamic evidence for Golgi apparatus involvement in biosynthesis came from light microscope radioautographic studies by Jennings and Florey in the 1950's, in which label was localized to the supranuclear Golgi region of goblet cells soon after injection of ³⁵S-sulfate. When the low energy isotope tritium became available, and when radioautography could be extended to the electron microscope level, a great improvement in spatial resolution was achieved. Studies using ³H-amino acids revealed that proteins were synthesized in the rough endoplasmic reticulum, migrated to the Golgi apparatus, and thence to secretion granules, lysosomes, or the plasma membrane. The work of Neutra and Leblond in the 1960's using ³H-glucose provided dramatic evidence that the Golgi apparatus was involved in glycosylation. Work with ³H-mannose (a core sugar in N-linked side chains), showed that this sugar was incorporated into glycoproteins in the rough endoplasmic reticulum, providing the first radioautographic evidence that glycosylation of proteins did not occur solely in the Golgi apparatus. Studies with the tritiated precursors of fucose, galactose, and sialic acid, on the other hand, showed that these terminal sugars are mainly added in the Golgi apparatus. With its limited spatial resolution, radioautography cannot discriminate between label in adjacent Golgi saccules. Nonetheless, in some cell types, radioautographic evidence (along with cytochemical and cell fractionation data) has indicated that the Golgi is subcompartmentalized in terms of glycosylation, with galactose and sialic acid being added to glycoproteins only within the trans-Golgi compartment. In the last ten years, radioautographic tracing of radioiodinated plasma membrane molecules has indicated a substantial recycling of such molecules to the Golgi apparatus.     

Using the scanning electron microprobe and secondary ion mass spectrometry to locate 14C- and 13C-labelled plant residues within soil aggregates

Increasing concentrations of CO2 in the atmosphere are placing emphasis on the necessity for sequestering carbon (C) into soil organic matter (SOM). By studying the interior parts of soil aggregates, a better understanding of the incorporation and sequestration of plant residue materials within these aggregates could be obtained. The location of newly added plant residues within soil aggregates may also assist in the investigation of the impact of these newly added plant materials on soil aggregation. This study investigated two different techniques for determining the location of newly added plant residues within soil aggregates by using plant materials labelled with 14C and 13C isotopes incorporated into two different soil types, Black Earth (Pellic Vertisol) and Red Clay (Chromic Vertisol). Both autoradiography combined with scanning electron microprobe analysis (14C) and secondary ion mass spectrometry (SIMS) (13C) were successfully used for detecting the presence and location of the newly added plant residues fragments within soil aggregates of both soil types. The use of labelled plant materials is essential for the study of the location of newly added plant materials within soil aggregates, and this has proven to be a useful tool for studying the impact of residue additions on soil aggregate formation. Furthermore, these methods have been shown to be useful for determining the incorporation and sequestration of C materials within soil aggregates. The development of the 13C SIMS technique could alleviate the necessity for the use of the radioactive isotope 14C in soil studies.     

Using the scanning electron microprobe and secondary ion mass spectrometry to locate 14C and 13C labelled plant residues within soil aggregates

Increasing concentrations of CO2 in the atmosphere are placing emphasis on the necessity for sequestering carbon (C) into soil organic matter (SOM). By studying the interior parts of soil aggregates, a better understanding of the incorporation and sequestration of plant residue materials within these aggregates could be obtained. The location of newly added plant residues within soil aggregates may also assist in the investigation of the impact of these newly added plant materials on soil aggregation. This study investigated two different techniques for determining the location of newly added plant residues within soil aggregates by using plant materials labelled with 14C and 13C isotopes incorporated into two different soil types, Black Earth (Pellic Vertisol) and Red Clay (Chromic Vertisol). Both autoradiography combined with scanning electron microprobe analysis (14C) and secondary ion mass spectrometry (SIMS) (13C) were successfully used for detecting the presence and location of the newly added plant residues fragments within soil aggregates of both soil types. The use of labelled plant materials is essential for the study of the location of newly added plant materials within soil aggregates, and this has proven to be a useful tool for studying the impact of residue additions on soil aggregate formation. Furthermore, these methods have been shown to be useful for determining the incorporation and sequestration of C materials within soil aggregates. The development of the 13C SIMS technique could alleviate the necessity for the use of the radioactive isotope 14C in soil studies.     

Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2-14%), myoviruses were moderately susceptible to tail breakage (15-40%), and siphoviruses were highly susceptible (32-76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. II. Other preparative methods

The loss of ¹⁴C-ethanolamine- and ³H-choline-labelled phospholipids from rat liver during preparation for electron microscopy by some less frequently used processing methods has been examined. Permanganate and formaldehyde-potassium dichromate fixation followed by Araldite embedding were investigated and five procedures involving embedding in water-miscible methacrylates (GMA). These procedures included a conventional method of dehydration and embedding in GMA, a low-temperature GMA embedding method, dehydration with ethylene glycol, freeze drying and freeze substitution. These results are compared with those obtained after conventional tissue preparation (presented previously, Cope & Williams, 1969). Formaldehyde-potassium dichromate compared favourably with the conventional procedures for the preservation of both phosphatides, especially phosphatidyl ethanolamine. Permanganate fixation was much less effective. Severe loss of both phosphatides occured after freeze drying and freeze substitution in glutaraldehyde-alcohol. GMA is shown to be a more potent phospholipid solvent than ethanol under the conditions employed. Low-temperature embedding reduced the loss of phosphatidyl choline during embedding. Results obtained by scintillation counting were confirmed by grain counts on thick-section autoradiographs. No direct relationship between extraction and the electron-microscopic appearance of membranes was discernible. It is believed that membrane prominence is largely dependent upon the electron density of the surrounding cytoplasm rather than on the degree of phospholipid extraction.     

Proliferative characteristics of epithelial cells in the pregnant rat uterus

In the pregnant rat, killed at about mid gestation and 1 h after injection of tritiated thymidine, 40% of the cells in the epithelium lining the uterine lumen at the implantation site were labelled. Between implantation sites fewer than 20% of the surface epithelial cells were labelled. A series of rats was given tritiated thymidine on day 12 of pregnancy and killed at intervals in the next 30 h. A percentage labelled mitoses analysis of the epithelium between implantation sites (interconceptual) and within the implantation site (conceptual) showed that cells in either region spent 7 h in DNA synthesis and 1.5 h in the G₂ + ½ mitosis phases. The epithelial cells in the conceptual region spent 1.5 h in the G₁ + ½ mitosis phases whereas cells in interconceptual regions spent at least 11.5 h in these phases. The average cycle times of cells in conceptual regions was 10 h: in interconceptual regions minimum cycle time was 20 h and the average appeared to be considerably longer. The grain count of the epithelial cells in the conceptual region was rapidly reduced during the 30 h after injection of tritiated thymidine suggesting successive rounds of cell division. In contrast the grain count distribution of cells in interconceptual regions changed only slowly during this time. The percentage of labelled epithelial cells was determined in the animals killed up to 30 h after injection of tritiated thymidine. In both conceptual and interconceptual regions these percentages increased initially as labelled cells produced labelled progeny. In the conceptual region the increase was not maintained after 7 h as cells initially in G₁ divided to give unlabelled progeny. In the interconceptual region the increase in the percentage of labelled cells continued for 14 h; thereafter the percentage did not significantly alter. The interpretation of these results is discussed in relation to the differences in the kinetic characteristics of the epithelial cells in the two regions and in relation to the morphology of the epithelium lining the uterus during pregnancy.     

基于液相色谱-质谱的血清糖蛋白质组定量分析

糖尿病作为最常见的代谢疾病之一，是一种受环境和遗传因素影响的多因素疾病。糖基化可导致蛋白功能多样性，对重大疾病、免疫系统有着深远影响。本实验通过两性离子亲水相互作用色谱(zwitterionic hydrophilic interaction liquid chromatography, ZIC-HILIC)富集技术结合微升级高效液相色谱-四极杆飞行时间质谱法，对6例糖尿病人和6例正常人的血清中的蛋白和糖蛋白进行了非标记定量分析，定量了65个差异蛋白和24个差异表达的糖蛋白。通过DAVID软件对这些差异蛋白和差异糖蛋白进行功能富集分析，进一步了解它们的细胞定位、分子功能和生物过程。该研究揭示了糖尿病发病过程中糖蛋白的变化，可为疾病的发病机制和临床诊断提供数据参考。     

反相液相色谱-电喷雾-离子阱-飞行时间质谱法定量分析N-非取代肝素/硫酸乙酰肝素

肝素和硫酸乙酰肝素由重复的二糖单元组成,其组分和结构与重要的生物病理、生理作用息息相关。这些二糖单元包括含量较高的N-硫酸化葡萄糖胺、N-乙酰化葡萄糖胺和含量较低的N-非取代葡萄糖胺残基。本研究采用2-氨基吖啶酮标记二糖,结合反相液相色谱-电喷雾-离子阱-飞行时间质谱法(RP-LC-ESI-IT-TOF MS)分析肝素/硫酸乙酰肝素中N-非取代二糖。通过优化检测条件,实现了12种肝素/硫酸乙酰肝素二糖的基线分离,并采用外标法相对定量分析各二糖组分。该方法适用于分析含有N-非取代二糖的肝素衍生物,可为进一步研究N-非取代葡萄糖胺的结构与功能提供检测方法,有助于更好地理解N-非取代葡萄糖胺残基在人类健康与疾病管理中的重要作用。     

双目视觉测量中等匹配点的光条中心提取

为了精确、快速提取激光条中心,使其提取结果更适用于工业现场特征尺寸的双目视觉测量,提出了一种等匹配点的激光条中心提取方法。利用灰度重心法粗提取出激光条中心点,计算灰度梯度方向,确定光条边界。接着,根据左右图像中激光条的粗提取结果确定基准光条,对另一幅图像中的对应光条进行插值。然后结合灰度梯度方向与插值结果对激光条进行重提取,得到等匹配点的亚像素中心坐标。最后,利用激光条中心点提取结果重建陶瓷平板、金属板表面及加工现场锻件表面的特征信息。实验结果表明,本文方法提取陶瓷板与金属板的激光条中心点的匹配率分别为99.887%与98.276%,宽度重建的相对误差分别为0.638%与0.488%,激光条中心提取结果能有效重建锻件表面的特征信息,满足工业现场对测量的精度、速度和鲁棒性要求。     

Three-dimensional reconstruction of a plant dictyosome from series of ultrathin sections using computer image processing

A single dictyosome from an actively secreting ovary gland cell of Aptenia cordifolia has been reconstructed in 3-D from a series of twenty-nine electron micrographs by computer image processing. The reconstruction is presented under different viewing angles in the form of shaded perspective displays. From these displays the entire dictyosome, surrounded by numerous vesicles, appears to be more a spherical than a flat body. The plate-like region of the dictyósome is demonstrated when only a portion of the electron micrographs is used for the image processing, leading to ‘cut-off’ displays. Since some upper planes were removed, such ‘cut-off’ displays revealed both tubular connections between cisternae of the dictyosome and the neighbouring endoplasmic reticulum as well as tubular continuities between adjacent Golgi cisternae within the same stack. Possible consequences of both types of interconnections on transport and processing of proteins and glycoproteins are discussed.     

Dense nuclei segmentation based on graph cut and convexity–concavity analysis

With the rapid advancement of 3D confocal imaging technology, more and more 3D cellular images will be available. However, robust and automatic extraction of nuclei shape may be hindered by a highly cluttered environment, as for example, in fly eye tissues. In this paper, we present a novel and efficient nuclei segmentation algorithm based on the combination of graph cut and convex shape assumption. The main characteristic of the algorithm is that it segments nuclei foreground using a graph‐cut algorithm with our proposed new initialization method and splits overlapping or touching cell nuclei by simple convexity and concavity analysis. Experimental results show that the proposed algorithm can segment complicated nuclei clumps effectively in our fluorescent fruit fly eye images. Evaluation on a public hand‐labelled 2D benchmark demonstrates substantial quantitative improvement over other methods. For example, the proposed method achieves a 3.2 Hausdorff distance decrease and a 1.8 decrease in the merged nuclei error per slice.     

An evaluation of various double labelling and autoradiographic techniques for the measurement of DNA synthesis time in leukaemic cells

Before applying the double labelling technique to the measurement of DNA synthesis time in leukaemic cells, various methods of labelling and of autoradiography were compared. The use of low and high doses of ³H-thymidine proved to be impracticable because, owing to a wide range of uptake per cell, it was impossible to make a clear distinction between ‘lightly’ and ‘heavily’ labelled cells. Two consecutive doses of ³H-thymidine resulted in only a small increase in labelling index because of the low percentage of cells in the S-phase and the long duration of DNA synthesis. Consequently, it was not possible to establish the DNA synthesis time with any degree of accuracy by this method. The remaining method employs ³H-thymidine and ¹⁴C-thymidine. Auto-radiographs made with a single emulsion proved difficult to interpret since some ¹⁴C-labelled cells give the appearance of being labelled with ³H-thymidine only. A double emulsion autoradiographic technique is to be preferred, but even then, more than one interval between the two isotopes is necessary in order to eliminate an error in the recognition of ¹⁴C-labelled cells.     

液相色谱-串联质谱法测定猪肝中痕量己烯雌酚、己烷雌酚和双烯雌酚

建立了液相色谱-串联质谱法测定猪肝中痕量己烯雌酚、己烷雌酚和双烯雌酚。以氟化铵为流动相添加剂,目标物响应值平均提高约10倍,向样品中加入1%(V/V)乙酸乙腈超声提取,提取液经分散固相萃取净化、水稀释后,采用Waters Acquity UPLC HSS T3色谱柱,以0.1 mmol/L氟化铵和乙腈为流动相,梯度洗脱分离,电喷雾电离源负离子模式和多反应监测模式检测,外标法定量。在0.4、0.8、4.0μg/kg添加水平下,3种目标物的加标回收率为89.5%～109.4%,相对标准偏差(RSD)为2.2%～7.8%,方法定量限为0.4μg/kg。本方法前处理过程无需浓缩,操作简便、灵敏度高、重现性好、环保节约,适用于猪肝中痕量己烯雌酚、己烷雌酚和双烯雌酚的定性与定量分析。     

HPLC-MS/MS法快速测定淀粉生物粘附材料特异性结合糖类物质的含量

研究了高效液相色谱-串联质谱(HPLC-MS/MS)法测定淀粉生物粘附材料与糖残基特异性结合实验中,岩藻糖、半乳糖、α-甲基甘露糖苷和N-乙酰葡萄糖胺4种糖类物质含量的分析方法。将反应后的样品直接离心过滤后即可利用HPLC-MS/MS进行定性定量分析。以V(乙腈)∶V(水)=80∶20的溶液作为液相色谱流动相,质谱采用电喷雾负离子电离方式扫描,以多反应监测(MRM)模式进行定量分析。在添加水平为1.0~40.0 mg.L-1范围内,岩藻糖的回收率为111.0%,相对标准偏差(RSD)为6.55%;在添加水平为1.0~80.0 mg.L-1范围内,半乳糖、α-甲基甘露糖苷和N-乙酰葡萄糖胺的回收率分别为106.5%、99.0%和91.0%,相对标准偏差分别为7.78%、6.64%和5.68%。4种糖类物质的方法检出限(S/N≥3)分别为0.1mg.L-1(岩藻糖),1.0 mg.L-1(半乳糖),0.1 mg.L-1(α-甲基甘露糖苷)和0.5 mg.L-1(N-乙酰葡萄糖胺)。该方法可用于评价淀粉粘附材料和糖类物质的特异性结合,为其他领域中糖类物质含量的测定提供可借鉴的方法。     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. I. Glutaraldehyde, osmium tetroxide, Araldite methods

The loss of ¹⁴C ethanolamine- and ³H choline-labelled phospholipids from rat liver during tissue preparation for electron microscopy has been examined. Column and thin-layer chromatography combined with double-label scintillation spectrometry were used to analyse the radioactive phospholipid content of the livers of rats injected simultaneously with ¹⁴C aminoethanol and ³H choline chloride. After 4 h (in vivo) the ¹⁴C and ³H labels were mainly incorporated into phosphatidyl ethanolamine and phosphatidyl choline respectively but some ¹⁴C label had been incorporated into phosphatidyl choline. Chopped rat liver was fixed in glutaraldehyde or osmium tetroxide or both sequentially and tissues were dehydrated in ethanol and embedded in Araldite. In each procedure examined the choline label proved more labile than the ethanolamine. After glutaraldehyde fixation alone complete loss of phosphatidyl choline occurred and half of the phosphatidyl ethanolamine was also lost. Following osmium tetroxide fixation negligible loss of either phosphatide occurred. In terms of phospholipid retention, no advantage was gained by glutaraldehyde fixation prior to osmium tetroxide fixation. The results show that both ethanols and embedding monomers are potent phospholipid solvents. The data also suggests that EM autoradiography of these two phosphatides may be carried out with reasonable confidence although it must be pointed out that a high degree of retention does not necessarily imply retention in situ.     

Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells

Confocal scanning laser microscopy has been used to make three-dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno-labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double-fluorescence emission requires an image processing step for the correction of ‘contamination’ effects due to the overlap between fluorescein and Texas Red emission spectra. At the pre-natal period (day 20 of gestation) each type of intermediate filament labelling is only present in a certain cellular category, C18 cytokeratin in hepatocytes and vimentin in mesenchymal cells. However, at the earliest developmental stages (day 12 of gestation), vimentin and cytokeratin seem to be found in the same type of cells, probably mesenchymal cells. Some striking developmental changes, associated with the differentiation of the liver parenchyma, are observed for both C18 cytokeratin and vimentin. In earlier foetal stages, C18 filaments are scarce, hazily labelled and randomly distributed inside the hepatocytic cytoplasm. Late during foetal development (days 18–20 of gestation), hepatocytic cytokeratin filaments are abundant, well individualized and sharply labelled. The hepatocytes are arranged in a muralium duplex architecture (two-cell-thick sheets) and the labelling intensity measured in the hepatocytic cytoplasm at the basal pole is double that measured at the sinusoidal pole, while, in the adult, hepatocytes are arranged in a muralium simplex architecture (one-cell-thick sheets) and cytokeratin filaments have a symmetrical distribution in relation to the nuclear region.     

西甜瓜中农药多残留方法研究

本研究采用了超高效液相色谱-串联质谱联用（UPLC-MS/MS）技术,建立了同时检测西甜瓜中14种农药的多残留检测方法。样品经乙腈提取,氨基固相萃取柱净化,Waters HSST3超高效液相色谱柱分离,进入电喷雾串联四级杆质谱进行检测,采用多反应监测（MRM）分析,对液质分离条件和样品前处理条件进行了优化。结果表明14种农药在10-200μg/L范围内线性良好（r≥0.9961）。在0.01、0.05、0.10mg/kg浓度范围内,平均加标回收率在80.1%-112.2%之间;相对标准偏差RSD（%）≤9.2%。该方法的最低检出限范围为1.92×10-4-2.38×10-3mg/kg。     

Capacitive measuring system for two-phase flow monitoring. Part 1: Hardware design and evaluation

Two-phase flows are commonly found in many industrial applications, such as oil and gas production. The monitoring of such flows is performed either in field applications or in pilot plant studies. In both cases, simple and robust measuring techniques are required. Capacitive probes have been applied for void fraction measurement in pipes in research and industry. However, capacitive measuring systems applied so far are tailored for specific applications and may not be easily adaptable. In addition, more and more soft-computing methods are applied for advanced data processing and parameter extraction which requires more computational power of sensor systems for online data processing. We develop a capacitive system provided with a microcontroller in which necessary routines for data processing may be embedded. System design is detailed explained and system's performance is evaluated, showing appropriate accuracy and time response for the investigation of two-phase flows.     

加速溶剂萃取法结合HPLC-ESI-TOF MS测定清肝散结汤中29种化学成分

清肝散结汤（QGSJ）是由14味不同中药组成的复方制剂，基于长期的实验观察和临床经验以不同组合成方用于肝癌的治疗。本研究建立了一种基于加速溶剂萃取（ASE）结合高效液相色谱-电喷雾飞行时间质谱（HPLC-ESI-TOF MS）的方法同时测定清肝散结汤中29种化学成分。使用70%乙醇作为提取溶剂，提取温度100 ℃，静态萃取时间5 min，提取2次，以上条件采用正交设计和主成分分析进行优化。HPLC分离采用Kromasil C18色谱柱，梯度洗脱。TOF MS在负离子模式下检测，质量扫描范围m/z 100~1100。结果表明：清肝散结汤中29种成分具有良好的线性关系（r＞0.994）和日内、日间精密度（RSD＜5%）；提取回收率在96.5%~104.5%之间。该方法快速、可靠，适用于复方中药的定量评价。