Steady State and Time Resolved Fluorescence Quenching and Chemical Modification Studies of a Lectin from Endophytic Fungus <Emphasis Type="Italic">Fusarium solani</Emphasis>

The solute quenching studies of a lectin from endophytic fungus Fusarium solani were carried out using different quenchers such as acrylamide, succinimide, potassium iodide and cesium chloride. The lectin showed emission maximum at 348 nm indicating relative exposure of tryptophan. The quenchable fraction of the fluorophore was 100% with acrylamide, whereas it was only 50% with succinimide. The ionic quenchers iodide and cesium showed opposite effects at different pH. In the case of cesium, raising the pH resulted in increased quenching and accessibility of typtophan residue, while the iodide showed just opposite effect. These studies showed that the single tryptophan residue of the lectin (per monomer) is relatively exposed, and might be in the vicinity of positively charged amino acid residues. Various amino acids of the F. solani lectin were modified using different reagents to obtain information about the hemagglutinating site. The chemical modification studies suggested tyrosine residues can be modified using N-acetylimidazole, which results in complete loss of hemagglutination activity of the lectin. Kinetics of chemical modification suggested involvement of only 2 tyrosine residues. Modification of arginine, cysteine, histidine, lysine, aspartate, glutamate and tryptophan did not result in loss of hemagglutinating activity of the lectin.     

Characterization of keyhole limpet hemocyanin (KLH) glycans sharing a carbohydrate epitope with Schistosoma mansoni glycoconjugates

Keyhole limpet hemocyanin (KLH) is known to share carbohydrate epitopes with Schistosoma mansoni. In order to define the structural basis for the observed serological cross-reactivity, KLH glycans were released either by enzyme treatment or by hydrazinolysis and probed with a rabbit hyperimmune serum directed against S. mansoni egg antigen. Both major, non-reacting oligosaccharide species as well as the minor compounds recognized were isolated by two-dimensional high performance liquid chromatography and in part by lectin affinity chromatography, and characterized by mass spectrometry. The results revealed that KLH carries predominantly high mannose-type glycans as well as short sugar chains. As a characteristic feature, a number of the latter glycans contained a Gal(beta1-6)Man-unit, which has not yet been found in glycoprotein-N-glycans. Oligosaccharides cross-reacting with schistosomal glycans comprised a terminal Fuc(alpha1-3)GalNAc-motif, which appears to represent the main carbohydrate epitope mediating cross-reactivity of KLH with glycoconjugates from S. mansoni.     

Structures and application of oligosaccharides in human milk

Comparative study of the oligosaccharide profiles of individual human milk revealed the presence of three different patterns. Four oligosaccharides containing the Fucα1-2Gal group were missing in the milk of non-secretor, and three oligosaccharides containing the Fucα1-4GlcNAc group were missing in the milk of Lewis negative individuals. Disappearance of some major oligosaccharides in these samples led to the finding of five novel minor oligosaccharides, which were hidden under the missing oligosaccharides. Following these studies, structures of many novel milk oligosaccharides were elucidated. At least 13 core oligosaccharides were found in these oligosaccharides. By adding α-fucosyl residues and sialic acid residues to these core oligosaccharides, more than one hundred oligosaccharides were formed. All these oligosaccharides contain lactose at their reducing termini. This evidence, together with the deletion phenomena found in the milk oligosaccharides of non-secretor and Lewis negative individuals, suggested that the oligosaccharides are formed from lactose by the concerted action of glycosyltransferases, which are responsible for elongation and branching of the Galβ1-4GlcNAc group in the sugar chains of glycoconjugates on the surface of epithelial cells. Therefore, oligosaccharides in human milk could include many structures, starting from the Galβ1-4GlcNAc group in the sugar chains of various glycoconjugates. Many lines of evidence recently indicated that virulent enteric bacteria and viruses start their infection by binding to particular sugar chains of glycoconjugates on the target cell surfaces. Therefore, milk oligosaccharides could be useful for developing drugs, which inhibit the infection of bacteria and viruses.     

Ultrastructural analysis and immunocytochemical localization of isolectins in Cratylia mollis seeds

Cratylia mollis is a native forage from the semi-arid region of Northeast, State of Pernambuco, Brazil, whose seeds have been considered an important lectin source. Multiple molecular forms of lectins, carbohydrate-binding proteins, have been purified from C. mollis seeds (Cra Iso) allowing several applications of these purified proteins. In this work seeds were processed for ultrastructural analysis and immunocytochemical localization of the two most abundant isolectins, Cra Iso 1 and Cra Iso 3, with glucose/mannose and galactose specificities, respectively. The ultrastructural analysis revealed a typical plant cell: organelles, nucleus and cellular wall were visualized. The localization of isolectins occurred mainly in the amorphous matrix of protein bodies, and in the cellular walls of the embryonic axis. The results showed that the isolectins, which differ in relation to carbohydrate specificity and glycosylation are located in the same cellular compartment suggesting different functions inside the same subcellular organelle. Cra Iso 1 and Cra Iso 3 distribution in the C. mollis seeds was consistent with the subcellular localization of several legume lectins.     

Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described.     

Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides

The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells. Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents. Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation. In the current study we hypothesize that a targeted search of sequence space surrounding the antigen-binding consensus sequence will select unpredictable amino acid sequences in the non-consensus portions of the peptides, leading to increased affinity for the carbohydrate and greater solubility in physiological buffers. This comprehensive in vitro analysis demonstrates that preferential evolution of the amino-terminal sequence of the peptides occurred, which correlated, in structure/function studies, with the acquisition of maturated function. The maturated peptides are more soluble than the earlier peptides. Studies of peptide binding to the disaccharide indicate that two maturated peptides (P-30-1, F03) have higher affinity for the antigen and bind with higher intensity to the surface of cultured human carcinoma cells than the first-generation peptides. The results support our hypothesis that affinity maturation can improve carbohydrate binding by peptides and have theoretical importance as the first report of maturation of carbohydrate-binding affinity in a small, soluble peptide.     

基于热图像的种蛋气室变化监测算法

种蛋气室的大小是监测种蛋孵化过程的重要指标之一。根据种蛋的热力学结构,种蛋在孵化过程中,包裹气室部分蛋壳会与其他部分蛋壳产生温差,从而可通过热红外图像进行观察。针对在种蛋孵化过程中,人工照蛋检测气室效率低的问题,探索设计了一种基于热图像的种蛋气室变化俯视监测算法。监测种蛋气室热图像的算法主要包括种蛋目标检测,种蛋图像分割和种蛋气室面积计算3个部分,其中种蛋的目标检测采用Faster-RCNN算法实现;种蛋图像分割采用BP神经网络算法实现;种蛋气室面积是在种蛋图像分割的基础上进行计算。使用孵化5天及以上的种蛋作为研究对象,并拍取种蛋的热图像进行试验。试验结果表明：种蛋热图像的目标检测的平均精度（mAP）为99.85%,拥有较好的检测效果。使用BP网络对种蛋进行图像分割。BP神经网络经过调参后,其网络最佳的结构为三层隐藏层,每个隐藏层拥有1 000个神经元,最优初始学习率为0.000 1,最优最大迭代次数为500。以F1-measure作为分割效果的评价指标,BP神经网络的图像分割总体结果为87.02%,Otsu算法的总体结果为65.25%。其中只有一个蛋的情况下,BP神经网络的分割结果为87.17%,Otsu算法的结果为68.86%。存在其他种蛋的干扰条件下,BP神经网络的分割结果为86.94%,Otsu算法的结果为61.64%,BP神经网络的分割效果优于Otsu分割算法,BP神经网络拥有更强的抗干扰能力。最后提取了孵化5～19 d种蛋的气室变化,通过观察种蛋气室大小曲线来监测种蛋的孵化情况,可看出随着天数的增加,气室有着明显变大的趋势。人工测量法与热红外测量法比较结果说明两者相关性为0.934 3,拥有较好的相关性。基于热图像的种蛋气室变化监测算法可在实际生产中实现种蛋的识别与气室大小的快速监测,为实现监测种蛋孵化的自动化提供了技术参考。     

Kinetics and Thermodynamics of Glycans and Glycoproteins Binding to Holothuria scabra Lectin: A Fluorescence and Surface Plasmon Resonance Spectroscopic Study

Holothuria scabra produces a monomeric lectin (HSL) of 182 kDa. HSL showed strong antibacterial activity and induced bacterial agglutination under in vitro conditions, indicating its role in animals’ innate immune responses. Very few lectins have been reported from echinoderms and none of these lectins have been explored in detail for their sugar-binding kinetics. Affinity, kinetics and thermodynamic analysis of glycans and glycoproteins binding to HSL were studied by fluorescence and surface plasmon resonance spectroscopy. Lectin binds with higher affinity to O-linked than N-linked asialo glycans, and the affinities were relatively higher than that for sialated glycans and glycoproteins. T-antigen α-methyl glycoside was the most potent ligand having the highest affinity (Ka 8.32 ×10⁷ M^?1). Thermodynamic and kinetic analysis indicated that the binding of galactosyl Tn-antigen and asialo glycans is accompanied by an enthalpic contribution in addition to higher association rate coupled by low activation energy for the association process. Presence of sialic acid or protein matrix inhibits binding. Higher affinity of HSL for O-glycans than N-glycans had biological implications; since HSL specifically recognizes bacteria, which have mucin or O-glycan cognate on their cell surfaces and play a major role in animal innate immunity. Since, HSL had higher affinity to T-antigen, makes it a useful tool for cancer diagnostic purpose.     

Comparative Studies of Two Araceous Lectins by Steady State and Time-Resolved Fluorescence and CD Spectroscopy

Transitions in the tryptophan microenvironment and secondary structure of two monocot lectins from Sauromatum guttatum and Arisaema tortuosum under different denaturing conditions were studied by steady state and time resolved fluorescence and CD spectroscopy. The lectins exist as tetramers with a single tryptophan residue estimated per monomer, present in a polar environment. Quenching with ionic quenchers showed predominantly electropositive environment for tryptophan residues. Acrylamide had maximum quenching effect. A decrease in KI quenching due to lectin denaturation indicated redistribution of charges as a result of possible conformational change. The two values for lifetimes of tryptophanyl population (1.2–1.4 and 6.3–6.4 ns) reduced substantially on quenching or denaturation. Similarly, both the lectins showed a drastic loss of secondary structure in 5 M Gdn-HCl or 6 M Urea or at pH 2.0 and below. For the first time araceous lectins, like legume lectins are shown to bind adenine. The presence of a compact structure at alkaline pH 10.0–12.0 was observed in CD spectra.     

Secretion of 13C-labelled oligosaccharides into human milk and infant's urine after an oral [13C]galactose load

Human milk oligosaccharides seem to play an important role in the infant's defense against bacterial and viral infections of the gastrointestinal and the urogenital tract. In this study, we investigated the influence of dietary carbohydrates on the biosynthesis of lactose and oligosaccharides in the human mammary gland and their renal excretion by the human milk-fed infant. For this purpose, a lactating woman was given 27 g galactose (Gal) containing 2 g [13C] Gal (1-13C/99%) immediately after breakfast. In the following 36 h, milk (5-10 ml) was collected before each nursing. Infant's urine was collected over a period of 24 h. 13C-enrichment was measured in total milk, milk fat and protein, in the carbohydrate fraction as well as in urine by isotope ratio mass spectrometry (IRMS). Milk carbohydrates and deproteinized urine samples were fractionated by Sephadex G25 gel filtration and further analyzed by IRMS, high performance thin layer chromatography and and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). IRMS revealed that in milk a maximal delta 13CPDB was reached within 8 h after Gal intake which then rapidly declined in the following 8 h. The cumulative 13C-elimination over this first peak was 6.9% of the oral 13C-dose. The highest 13C-enrichment was detectable in the carbohydrate fraction, mainly in lactose and neutral oligosaccharides. Compared to the enrichment of human milk, the delta 13CPDB of infant's urine was delayed. In urine, the highest amount of 13C was found in the Sephadex G25 fractions which mainly contained lactose, fucosyl-lactose, lacto-N-tetraose (LNT), fucosyl-LNT and difucosyl-LNT. For further characterization, individual components were separated by HPAEC-PAD and subsequently analyzed by fast atom bombardment mass spectrometry and IRMS. The data show, that orally applied Gal is incorporated in milk, especially in lactose and neutral oligosaccharides. Obviously, some of these components were absorbed by the infant and then excreted with urine. There, oligosaccharides may serve as analogous receptors for bacterial or viral adhesion molecules, and, hence, may prevent urogenital infections in breastfed infants.     

克雷伯氏肺炎杆菌内抗亚碲酸盐蛋白质TerZ 延伸环降低对钙离子亲合性

亚碲酸盐是碲的一含氧阴离子,其对微生物具高度毒性．在许多的致病菌内已经鉴定出数个抗亚碲酸盐基因(terZABCDEF)．之前,作者解出抗亚碲酸盐蛋白质TerD液体核磁共振结构并指出在细菌内TerD 可能是一钙离子传感器．TerZ 与TerD 在序列上有40%相同性,其包括了一额外的9 氨基酸片段L36-N44,并且显示出非常弱的钙离子亲合性．有趣的是,少了额外片段的TerZdel 拥有与TerD 可比较的钙离子亲合性．根据化学位移指数及同源模拟结果,此额外片段为一无二级结构且延伸的loop,可能扰乱钙离子结合位置的构形,同时也阻碍了钙离子接近其结合位置,因此大大降低钙离子亲合性．     

Critical parameters for the analysis of anionic oligosaccharides by desorption electrospray ionization mass spectrometry

Sulfated oligosaccharides derived from glycosaminoglycans (GAGs) are fragile compounds, highly polar and anionic. We report here on the rare but successful application of desorption electrospray ionization (DESI) - LTQ-Orbitrap mass spectrometry (MS) to the high-resolution analysis of anionic and sulfated oligosaccharides derived from the GAGs hyaluronic acid and heparin. For that purpose, key parameters affecting DESI performance, comprising the geometric parameters of the DESI source, the probed surface and the spraying conditions, applied spray voltage, flow rates and solvent composition were investigated. Under suitable conditions, the DESI technique allows the preservation of the structural integrity of such fragile compounds. DESI enabled the sensitive detection of anionic hyaluronic acid and heparin oligosaccharides with a limit of detection (LOD) down to 5 fmol (≈10?pg) for the hyaluronic acid decasaccharide. Detection of hyaluronic acid oligosaccharides in urine sample was also successfully achieved with LOD values inferior to the ng range. Multistage tandem mass spectrometry (MS(n) ) through the combination of the DESI source with a hybrid linear ion trap-orbitrap mass spectrometer allowed the discrimination of isomeric sulfated oligosaccharides and the sequence determination of a hyaluronic acid decasaccharide. These results open promising ways in glycomic and glycobiology fields where structure-activity relationships of bioactive carbohydrates are currently questioned. Copyright ? 2012 John Wiley & Sons, Ltd.     

Functional glycomics and evidence for gain- and loss-of-functions of target proteins for glycosyltransferases involved in N-glycan biosynthesis: their pivotal roles in growth and development,cancer metastasis and antibody therapy against cancer

N-Acetylglucosaminyltransferase V (GnT-V), N-acetylglucosaminyltransferase III (GnT-III) and α1-6 fucosyltransferase (Fut8) catalyze reactions that form biologically important branching N-linked sugar chains in glycoproteins. The above three branching N-glycan sugar chains, β1-6 GlcNAc branching, bisecting GlcNAc and core fucose (α1-6 fucose), play major roles in cancer invasion and metastasis, the inhibition of cancer metastasis, and antibody-dependent cellular cytotoxicity (ADCC), growth and development, respectively. A functional glycomic approach identified the gain- and loss-of-functions of glycoproteins as the result of the aberrant glycosylation. A membrane-type metal dependent serine proteinase designated matriptase which contains β1-6 GlcNAc branching became resistant to auto-digestion and proteolysis by trypsin, and resulted in a constitutively active form which might be implicated in cancer invasion and metastasis. GnT-V also acts as an angiogenic factor without the mediation of functions as a glycosyltransferase. Recently, a GnT-V homologue, GnT-IX has been identified. This gene is expressed at relatively high levels in the brain and acts on N-glycans to form a unique branched structure, as well as O-mannosyl glycans. The addition of bisecting GlcNAc to various signaling molecules or adhesion molecules suppresses cancer metastasis. Fut8 knock-out mice, due to the lack of a core fucose (α1-6 fucose) in target glycoproteins, show disorders in growth and development. The presence of a bisecting GlcNAc or the absence of a core fucose in IgG molecules enhances ADCC activity for killing tumor cells by up to 10 to 100 fold and therefore is thought to have considerable use in antibody therapy against cancer. These data clearly indicate that gain- and loss-of-functions of target proteins for these glycosyltransferases are biologically important.     

表面增强拉曼光谱检测鸡蛋蛋清内三聚氰胺的定量方法研究

三聚氰胺对人体有害,鸡蛋内三聚氰胺定量检测非常有必要。以鸡蛋蛋清为研究对象,应用表面增强拉曼光谱技术结合化学计量学方法对蛋清内三聚氰胺进行了定量检测。首先采用人工饲养蛋鸡的方法获取含有三聚氰胺的样品鸡蛋。然后使用便携式拉曼光谱检测仪(Opto Trace RamTracer-200)和拉曼增强试剂测定蛋清的表面增强拉曼光谱,同时利用气相色谱质谱技术测定相应蛋清中三聚氰胺的含量。利用Raman Analyzer对拉曼光谱基线进行校正。应用相关系数法从表面增强拉曼光谱中选取320个光谱变量作为输入变量,建立偏最小二乘定量校正模型;并应用谱峰分解法建立谱峰分解定量校正模型。两种模型建立过程中均选定90个样本做为模型校正集,44个样本做为模型验证集,两种模型都有较好的预测效果。偏最小二乘定量校正模型预测值与气相色谱质谱联用法(GC-MS)测定值的决定系数R2为0.856,预测均方根误差RMSEP为1.547;谱峰分解定量校正模型R2为0.947,RMSEP为0.893。实验结果表明,该方法能有效定量检测鸡蛋内三聚氰胺,检测一个样本仅需15 min,为蛋品的三聚氰胺检测提供了一种新途径。     

基于红外光谱的鲜地黄、生地黄和熟地黄成分差异分析

运用傅里叶变换衰减全反射红外光谱(ATR-FTIR)对鲜地黄水提物、生地黄水煎液和熟地黄水煎液的整体成分进行差异分析,并运用离子色谱法对差异成分进行定量分析,研究鲜地黄与其炮制品的整体成分及变化规律。ATR-FTIR中,三种地黄在糖区有差异,其中鲜地黄糖区特征峰在1 140, 1 047和1 000 cm^-1,生地黄糖区特征峰为1 140和1 045 cm^-1,熟地黄糖区特征峰为1 142和1 029 cm^-1,且三种地黄峰形状不同。二阶导数红外光谱(SDIR)进一步发现,地黄炮制过程中糖类成分在1 200～600 cm^-1之间的特征峰变化最为显著。三种地黄提取液的特征峰位置相近,但相对峰强度不同,且随着炮制过程呈规律性变化。可以推测出在鲜地黄炮制过程中,鲜地黄中多糖发生水解,变成熟地黄中的寡糖或者单糖。应用离子色谱法对8种单糖和寡糖进行定量分析,所建立方法线性关系良好(R²≥0.999 0)。定量结果显示,葡萄糖、蜜二糖、半乳糖、甘露糖、甘露三糖、水苏糖和毛蕊花糖苷在鲜地黄、...     

Plumbagin,a Naturally Occurring Naphthoquinone: Its Isolation,Spectrophotometric Determination in Roots,Stems, and Leaves in Plumbago Europaea L.

A new method for isolation and spectrophotometric determination of plumbagin is presented. Plumbagin was isolated by thin layer chromatography (TLC) and column chromatography (CC) techniques, as an orange tinged yellow long crystalline substances. Plumbagin exhibits two absrop-tion maxima at 410 and 510 nm. Stability of the color, pK_a value, and the effect of pH were studied. Beer's law is obeyed over the range 0.9-45 ppm. The method is applied to the determination of plumbagin in roots, stems, and leaves of Plumbago europaea L. plant.     

Ultrasonic-assisted enzymatic improvement of polyphenol content,antioxidant potential,and in vitro inhibitory effect on digestive enzymes of Miang extracts

The aims of this research were to optimize the ultrasonic-assisted enzymatic extraction of polyphenols under Miang and tannase treatment conditions for the improvement of antioxidant activity of Miang extracts via response surface methodology. Miang extracts treated with and without tannase were investigated for their inhibitory effects on digestive enzymes. The optimal conditions for ultrasonic-assisted enzymatic extraction of the highest total polyphenol (TP) (136.91 mg GAE/g dw) and total flavonoid (TF) (5.38 mg QE/g dw) contents were as follows: 1 U/g cellulase, 1 U/g xylanase, 1 U/g pectinase, temperature (74 °C), and time (45 min). The antioxidant activity of this extract was enhanced by the addition of tannase obtained from Sporidiobolus ruineniae A45.2 undergoing ultrasonic treatment and under optimal conditions (360 mU/g dw, 51 °C for 25 min). The ultrasonic-assisted enzymatic extraction selectively promoted the extraction of gallated catechins from Miang. Tannase treatment improved the ABTS and DPPH radical scavenging activities of untreated Miang extracts by 1.3 times. The treated Miang extracts possessed higher IC₅₀ values for porcine pancreatic α-amylase inhibitory activity than those that were untreated. However, it expressed approximately 3 times lower IC₅₀ values for porcine pancreatic lipase (PPL) inhibitory activity indicating a marked improvement in inhibitory activity. The molecular docking results support the contention that epigallocatechin, epicatechin, and catechin obtained via the biotransformation of the Miang extracts played a crucial role in the inhibitory activity of PPL. Overall, the tannase treated Miang extract could serve as a functional food and beneficial ingredient in medicinal products developed for obesity prevention.     

Xyloglucan degradation using different radiation sources: a comparative study

Tamarind seed xyloglucan was subjected to different radiation sources-ultrasound, gamma-radiation, and microwave heating, and the effects of these energies upon its molecular and structural properties were characterised by gel permeation chromatography, viscometry, sugar analysis, FT-IR and NMR spectroscopic techniques. In dependence on the degradation methods and experimental conditions used, the decrease of the relative molecular mass (RMM) was accompanied with alteration of the primary structure. Depolymerisation by ultrasound at a frequency of 20 kHz yielded after 120 min products with RMM of about 131 x 10(3) without significant alteration of the primary structure of the polysaccharide. Intense degradation of XG started by microwave heating at pH 1.5 yielding polymers with RMM in the range of higher oligosaccharides, however, with changed sugar composition due to cleavage of the glycosyl side chains. At gamma-irradiation doses >40 kGy, next to chain cleavage, very high-molecular mass components exhibiting UV(254)-absorption were formed, and the RMM decreased to about 50 x 10(3) at the highest applied dose (100 kGy). The results of the comparative study suggest that ultrasonication was the most convenient procedure to decrease the RMM of xyloglucan to 130 x 10(3) and preserve the primary structure of the polysaccharide.     

Determination of Bioactive Matter by Affinity Adsorption Solid Substrate–Room Temperature Phosphorimetry Based on Lectin Labeled with Self-ordered Ring of Eosin Y

A new phosphorescent labeling reagent named self-ordered ring (ESOR) of eosin Y (E) was developed. And the application of the determination of bioactive matter by affinity adsorption solid substrate–room temperature phosphorimetry (AA-SS-RTP) based on ESOR labeling lectin was studied. Results showed that pink and homogeneous ESOR could be formed by E on polyamide membrane (PAM) in the presence of cetyltrimethylammonium bromide (CTAB) and ammonia water. ESOR could emit strong and stable room temperature phosphorescence (RTP) signal of E in the presence of heavy atom perturber. Specific affinity adsorption (AA) reactions could be carried out between the products of concanavalin agglutinin (Con A), triticum vulgaris lectin (WGA) labeled with ESOR and alpha-fetoprotein variant (AFP-V), alkaline phosphatase (ALP), glucose (G), respectively. Not only did the products of the affinity adsorption reactions preserve good RTP characteristic of E, but also the ΔI _p (ΔI _p = I _p2 − I _p1, I _p1 is the RTP intensity of blank reagent, I _p2 is the RTP intensity of sample) of these products was proportional to the content of AFP-V, ALP and G, respectively. According to the facts above, a new method of AA-SS-RTP for the determination of AFP-V, ALP and G was established, based on ESOR labeling lectin. Detection limits (LD) of this method were 0.040 fg spot⁻¹ for AFP-V, 0.045 fg spot⁻¹ for ALP and 0.090 fg spot⁻¹ for G. And it has been successfully applied to the determination of AFP-V in human serum as well as the survey and forecast of human diseases. This method had high sensitivity, good repeatability, long RTP lifetime and little background interference with at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V by AA-SS-RTP based on Con A labeled with ESOR was also discussed.     

Acoustic detection and quantification of benthic egg beds of the squid Loligo opalescens in Monterey Bay, California

The squid Loligo opalescens is a key species in the nearshore pelagic community of California, supporting the most valuable state marine fishery, yet the stock biomass is unknown. In southern Monterey Bay, extensive beds occur on a flat, sandy bottom, water depths 20-60 m, thus sidescan sonar is a prima-facie candidate for use in rapid, synoptic, and noninvasive surveying. The present study describes development of an acoustic method to detect, identify, and quantify squid egg beds by means of high-frequency sidescan-sonar imagery. Verification of the method has been undertaken with a video camera carried on a remotely operated vehicle. It has been established that sidescan sonar images can be used to predict the presence or absence of squid egg beds. The lower size limit of detectability of an isolated egg bed is about 0.5 m with a 400-kHz sidescan sonar used with a 50-m range when towed at 3 knots. It is possible to estimate the abundance of eggs in a region of interest by computing the cumulative area covered by the egg beds according to the sidescan sonar image. In a selected quadrat one arc second on each side, the estimated number of eggs was 36.5 million.