We have studied in detail the effects of nitrogen implantation into a p + polysilicon gate on gate oxide properties for the surface p-channel metal oxide semiconductor (PMOS) below 0.25 µm. The nitrided oxide film can be easily formed by the pile-up of nitrogen into the gate oxide film from the polysilicon gate. It was found that boron penetration through the gate oxide film can be effectively suppressed by nitrogen implantation into a p + polysilicon gate because nitrogen in the polysilicon film can suppress boron diffusion, and the nitrided oxide film can also act as a barrier to boron diffusion. Moreover the hot-carrier hardness can be remarkably improved by the nitrided oxide film since interface state generation can be suppressed by the nitrided oxide film. Furthermore the number of electron traps in the gate oxide film can also be reduced by nitrogen implantation.
Threshold-voltage (Vth) variation of silicon on thin buried oxide (SOTB) complementary metal–oxide–semiconductor (CMOS) transistors and the impact of reducing the variation on leakage current were studied. Both reduction of impurity concentration in the silicon-on-insulator (SOI) layer and suppression of short-channel effect without the halo implantation were essential for reducing the Vth variation. Using a metal-gate was also effective. The standard deviation of Vth (σVth) for SOTB with fully silicided (FUSI) metal gate was half that for the bulk with the same gate size. This improvement can reduce the off-state leakage current summed over a large number of transistors by half in the 65-nm technology. With further scaling of the gate length, this effect can be enhanced. The SOTB of small σVth has a strong impact on reducing leakage current in highly scaled LSI.
A conversion process from potassium tetratitanate fibers into potassium hexatitanate was investigated to develop potassium hexatitanate fibers with negligible leachability which are usable as a filler for fiber-rein-forced polyester thermoplastic resins. Calcined potassium tetratitanate fibers were dispersed in water. By titrating the above mentioned suspension with an aqueous HCI solution to adjust the value of ([K+]/[H3O+])2/3=5.71×105 at 60°C, the fibers with TiO2/K2O=5.95 was obtained. The product thus obtained was heated at 1000°C for 30min, then potassium hexatitanate fibers having a tunnel structure were obtained. Then, the water soluble potassium was further removed with an aqueous HCI solution. Potassium hexatitanate fibers having a tunnel structure whose molar ratio of TiO2/K2O was 6.0 was obtainable. Water soluble potassium thus prepared was only 4 mass ppm. The potassium hexatitanate fibers prepared by the novel method developed by the present authors were found useful as a reinforcing material for polycarbonate which has been known to be vulnerable to soluble potassium.
An ultra-small RFID chip uses an electron beam for writing 1T memory cells. A 90nm SOI CMOS process and double-surface electrode chip structures enable the design of 0.05times0.05mm 2 and 5mum-thick RFID chips with small, low-cost and highly-reliable 128b ID-memory. The chip is verified at a carrier frequency of 2.45GHz with measured communication distance of 300mm.
The temperature coefficient of V th (= d V th / d T ), which is commonly utilized for circuit design, was systematically obtained against various TiN and capping layer thicknesses in high- k /metal gate field-effect transistors (FETs). It is known that the magnitude of | d V th / d T | for such FETs is larger than that of polycrystalline silicon (poly-Si) gate FETs. The origins of the d V th / d T difference among high- k /metal gate FETs were attributed to differences in the temperature coefficient of flat band voltage (= d V FB / d T ) and the equivalent gate oxide thickness (EOT). Thicker TiN layers reduced d V FB / d T , which enlarged the magnitude of | d V th / d T |. The EOT increased as the TiN metal layer or Al 2 O 3 capping layer increased in thickness. The large EOT led to an increase in | d V th / d T |, since d V th / d T is a function of the inverse of gate capacitance. In contrast, La 2 O 3 capping hardly affected d V th / d T . This is because La 2 O 3 capping did not affect EOT differently from Al 2 O 3 capping. The relationship between d V th / d T and EOT implies that EOT scaling relieves the issue of large | d V th / d T | for high- k /metal gate FETs.
More precise information on the degree of polymerization (DP) of polysialic acid (polySia) chains expressed on neural cell adhesion molecule (NCAM) and its developmental stage-dependent variation are considered important in understanding the mechanism of regulated polysialylation and fine-tuning of NCAM-mediated cell adhesion by polySia. In this paper, first we performed a kinetic study of acid-catalyzed hydrolysis of polySia and report our findings that (a) in (→8Neu5Acα2→)n→8Neu5Acα2→3Galβ1→R, the proximal Neu5Ac residue α2→3 linked to Gal is cleaved about 2.5–4 times faster than the α2→8 linkages and (b) in contrary to general belief that α2→8 linkages in polySia are extremely labile, the kinetic consideration showed that they are not so unstable, and every ketosidic bond is hydrolyzed at the same rate. These findings are the basis of our strategy for DP analysis of polySia on NCAM. Second, using the recently developed method that provides base-line resolution of oligo/polySia from DP 2 to >80 with detection thresholds of 1.4 fmol per resolved peak, we have determined the DP of polySia chains expressed in embryonic chicken brains at different developmental stages. Our results support the presence of numerous NCAM glycoforms differing in DPs of oligo/polySia chains and a delicate change in their distribution during development. More precise information on the degree of polymerization (DP) of polysialic acid (polySia) chains expressed on neural cell adhesion molecule (NCAM) and its developmental stage-dependent variation are considered important in understanding the mechanism of regulated polysialylation and fine-tuning of NCAM-mediated cell adhesion by polySia. In this paper, first we performed a kinetic study of acid-catalyzed hydrolysis of polySia and report our findings that (a) in (→8Neu5Acα2→)n→8Neu5Acα2→3Galβ1→R, the proximal Neu5Ac residue α2→3 linked to Gal is cleaved about 2.5–4 times faster than the α2→8 linkages and (b) in contrary to general belief that α2→8 linkages in polySia are extremely labile, the kinetic consideration showed that they are not so unstable, and every ketosidic bond is hydrolyzed at the same rate. These findings are the basis of our strategy for DP analysis of polySia on NCAM. Second, using the recently developed method that provides base-line resolution of oligo/polySia from DP 2 to >80 with detection thresholds of 1.4 fmol per resolved peak, we have determined the DP of polySia chains expressed in embryonic chicken brains at different developmental stages. Our results support the presence of numerous NCAM glycoforms differing in DPs of oligo/polySia chains and a delicate change in their distribution during development. neural cell adhesion molecule degree(s) of polymerization sialic acid N-acetylneuraminic acid or 2-keto-3,5-dideoxy-5-acetylamino-d-glycero-d-galacto-nononic acid N-glycolylneuraminic acid or 2-keto-3,5-dideoxy-5-glycolylamino-d-glycero-d-galacto-nononic acid α2→8-linked oligo/polyNeu5Ac 1,2-diamino-4,5-methylenedioxybenzene DMB-tagged α2,8-linked polyNeu5Ac with a DP n that is obtained by reacting (Neu5Ac)n+1 with DMB (Q denotes fluorescent chromophore, quinoxalinone derivative) high performance liquid chromatography high performance anion-exchange chromatography with pulsed electrochemical detection HPLC with fluorescence detection of ulosonates after derivatization with DMB reagent embryonic chicken at day 5 and day 10, respectively −logK a of ionization constant K a = [A−][H3O+]/[HA][H2O] for an equilibrium of HA + H2O ⇄ A− + H3O+ Polysialylation is a unique posttranslational modification of the neural cell adhesion molecule (NCAM),1 and polysialic acid (polySia) is a homopolymer of α2→8-linked Neu5Ac. PolyNeu5Ac chain is now known to regulate homophilic NCAM-NCAM adhesion and is shown to be crucial to synaptic plasticity in the developing and adult brain, thereby playing a critical role in various relevant pathophysiological events in vertebrates (see for example, Ref. 1Muhlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. Curr. Biol. 1996; 6: 1188-1191Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). PolySia is also known to occur primarily on NCAM in embryonic tissues, and if not exclusively, NCAM is a major carrier of this unique polymer in mammals. A large amount of data suggesting a complex, coordinated process of biochemically detectable changes in the molecule and morphological changes that govern the development of the nervous system are available, and the presence of distinguishable NCAM glycoforms differing in the content of Sia has been detected. Many of important biological and pathophysiological regulatory effects of the Sia content of NCAM on neural cellular events are governed by the degree of polymerization (DP) of polyNeu5Ac. However, until now the presence of the oligo/polyNeu5Ac residues in different glycoforms of NCAM has been only qualitatively referred to as different terminology in the literature as follows: (1) adult form (A-form) NCAM versusembryonic form (E-form) NCAM (the former contains only one-third of the Sia content of the latter) (2Hoffman S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5762-5766Crossref PubMed Scopus (517) Google Scholar, 3Schlosshauer B. Schwarz U. Rutishauser U. Nature. 1984; 310: 141-143Crossref PubMed Scopus (91) Google Scholar); (2) Sia-rich NCAM versusSia-poor form or NCAM with a lower Sia content (3Schlosshauer B. Schwarz U. Rutishauser U. Nature. 1984; 310: 141-143Crossref PubMed Scopus (91) Google Scholar, 4Friedlander D.R. Brackenbury R. Edelman G.M. J. 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Quantitative information on the length or DP versatility of polySia chains is considered important in understanding the molecular mechanism of biosynthesis of polySia chains and "coarse and fine tuning" of the adhesive behavior of the neural cells by a wide spectrum of glycoform structure of NCAM. Details of such information cannot be obtained using biochemical probes including anti-polySia antibodies and endo N-acylneuraminidase. We hypothesize that the possible presence of a continuum glycoform profile in certain functional glycoproteins is important in coarse and fine tuning of "protein-based functions," particularly in higher animals. Thus, the present study of determination of DP and distribution of oligo/polySia chains on NCAM represents one of the pilot studies for Glycome Project that complements the Genome-based Proteome Project. The present paper has for its objective the investigation of the length or DP of polySia chains on various stages of embryonic chicken brain using the recently developed ultrasensitive and selective method. To validate our strategy of DP determination of glycan chain-bound polySia, we carried out a kinetic study of acid-catalyzed hydrolysis of free oligo- and polySia with definite DP values and compared the stability of α2→8 sialosyl linkages with those of α2→3 and α2→6 sialosyl linkages in model compounds. From the analysis of the experimental results, it has been concluded that hydrolysis of all internal α2→8-linked sialosidic linkages in polySia takes place at the same rate, which is significantly slower than the sialosyl bond of the proximal Sia residue linked α2→3 to the Gal of the core glycan chain in NCAM, i.e. (Neu5Ac)n− 1Neu5Acα2→3Galβ1→ under the conditions used in this study. The results and interpretation of this study may argue against previous evidence of polySia chains that the DP of the polySia chain on NCAM is as large as or even larger than 100 (45Livingston B.D. Jacobs J.L. Glick M.C. Troy F.A. J. Biol. Chem. 1988; 263: 9443-9448Abstract Full Text PDF PubMed Google Scholar), and that polyNeu5Ac chains with high DP are extremely labile to hydrolytic breakdown even at near neutral pH (46Manzi A.E. Higa H.H. Diaz S. Varki A. J. Biol. Chem. 1994; 269: 23617-23624Abstract Full Text PDF PubMed Google Scholar). Neu5Ac dimer (Neu5Acα2→8Neu5Ac) was a gift from Nihon Gaishi (Handa, Japan). Neu5Acα2→3Galβ1→4Glc and Neu5Acα2→6Galβ1→4Glc were purchased from Glyko (Novato, CA). Colominic acid was purchased from Nacalai Tesque (Kyoto, Japan). Two polySia samples with discrete DP values, i.e. (Neu5Ac)10 and (Neu5Ac)21, and three other polyNeu5Ac samples of which the major components are (Neu5Ac)41, (Neu5Ac)67 + (Neu5Ac)68, and (Neu5Ac)76 were prepared by chromatographic separation of a controlled hydrolysate of commercially available colominic acid on a DEAE-Sephadex A-25 column (47Nomoto H. Iwasaki M. Endo T. Inoue S. Inoue Y. Matsumura G. Arch. Biochem. Biophys. 1982; 218: 335-341Crossref PubMed Scopus (82) Google Scholar) or by direct HPLC fractionation of commercial colominic acid without pre-hydrolysis on a MonoQ HR 10/10 (Amersham Pharmacia Biotech) or a DNAPac PA-100 (Dionex, Sunnyvale, CA) column. For some samples of (Neu5Ac)n the n values were determined by the HPAEC-PED method after pre-hydrolysis at 50 °C; this pre-hydrolysis was necessary to obtain a homologous series of peaks for (Neu5Ac)n, n = 1 ton, to determine n. For all samples examined,n values determined by the new DMB/HPLC-FD method were identical with those determined by HPAEC-PED. Total Neu5Ac was determined after maximal hydrolytic liberation of free Neu5Ac in 0.1m trifluoroacetic acid at 80 °C by reverse-phase HPLC of the DMB derivative of Neu5Ac as described previously (48Inoue S. Lin S.-L. Inoue Y. J. Biol. Chem. 2000; 275: 29968-29979Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) or by HPAEC-PED without derivatization (49Zhang Y. Inoue Y. Inoue S. Lee Y.C. Anal. Biochem. 1997; 250: 245-251Crossref PubMed Scopus (39) Google Scholar). Neu5Acα2→8Neu5Ac, Neu5Acα2→3Galβ1→4Glc, and Neu5Acα2→6Galβ1→4Glc (200 ng∼1 µg) were hydrolyzed in 0.02m trifluoroacetic acid at 50 °C or in 0.1 mtrifluoroacetic acid at 80 °C in total volume of 80 µl. The reaction was terminated by adding 20 µl of 0.2 m NaOH at appropriate time intervals. Two polySia samples with the discrete DP values, (→8Neu5Acα2→)10 and (→8Neu5Acα2→)21, were hydrolyzed in 0.02m trifluoroacetic acid at 50 °C. Mono- and oligo/polyNeu5Ac formed were quantified by HPAEC-PED (cf. Ref. 49Zhang Y. Inoue Y. Inoue S. Lee Y.C. Anal. Biochem. 1997; 250: 245-251Crossref PubMed Scopus (39) Google Scholar). Derivatization of polySia at the reducing end with the fluorogenic reagent DMB originally used for the labeling of Sia (50Hara S. Takemori Y. Yamaguchi M. Nakamura M. Ohkura Y. Anal. Biochem. 1987; 164: 138-145Crossref PubMed Scopus (297) Google Scholar) was carried out in the reaction mixture containing, finally, 2.7 m DMB (Dojinbo, Kumamoto, Japan), 9 mm sodium hydrosulfite, 0.5 mβ-mercaptoethanol, 20 mm trifluoroacetic acid, and a given Sia-containing reactant at 10 °C for 48 h. The reaction was stopped by adding one-fifth volume of 1 m NaOH to hydrolyze lactones formed during the reaction (51Cheng M.-C. Lin S.-L. Wu S.-H. Inoue S. Inoue Y. Anal. Biochem. 1998; 260: 154-159Crossref PubMed Scopus (36) Google Scholar, 52Zhang Y. Lee Y.C. J. Biol. Chem. 1999; 274: 6183-6189Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). For HPAEC-PED analysis, a DX-500 ion chromatography system (Dionex, Sunnyvale, CA) was used with an ED-40 electrochemical detector and a CarboPac PA-100 column. Elution of oligo/polySia was performed at 1 ml/min with a concentration gradient of NaNO3 as described previously, whereas the concentration of NaOH was always kept at 0.1 m (49Zhang Y. Inoue Y. Inoue S. Lee Y.C. Anal. Biochem. 1997; 250: 245-251Crossref PubMed Scopus (39) Google Scholar, 53Lin S.-L. Inoue Y. Inoue S. Glycobiology. 1999; 9: 807-814Crossref PubMed Scopus (29) Google Scholar). For DMB/HPLC-FD, a Hewlett-Packard HPLC system series 1100 was used with a DNAPac PA-100 column and a fluorescence detector set at 372 nm for excitation and 456 nm for emission (54Lin S.-L. Inoue S. Inoue Y. Carbohydr. Res. 2000; 329: 447-451Crossref PubMed Scopus (13) Google Scholar). Elution was performed at 1 ml/min with segments of linear gradient of NaNO3 made by introducing 1m NaNO3, 2, 2, 3, 10, 20, 25, and 35% in water at 0, 3, 6, 14, 28, 43, and 95 min, respectively. PolySia-containing glycopeptides were prepared and purified from embryonic chicken brain (E5∼E21) as described previously (48Inoue S. Lin S.-L. Inoue Y. J. Biol. Chem. 2000; 275: 29968-29979Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The samples were eluted under a slightly included peak earlier than commercial colominic acid (DP 100) from a Sephacryl S-200 column and under a peak eluted with higher concentrations of NaCl than for colominic acid from a MonoQ HR 10/10 column. Special attention was directed to the questions and assessment of the reliability and validity of the data obtained using a newly developed method of polySia analysis. To answer the question as to whether this DMB/HPLC-FD method can detect an original sample of polySia with high DP and give its correct DP value was most important because the derivatization was carried out under acidic conditions in which partial cleavage of inter-residue sialosyl linkages was unavoidable. For this purpose, we used polyNeu5Ac samples separated from colominic acid by anion-exchange chromatography, and the DP values of their major components were determined by HPAEC-PED (see "Experimental Procedures"). The chromatographic profiles obtained by the DMB/HPLC-FD method described under "Experimental Procedures" are given in Fig. 1, a–d. A series of lower DP fragments were detected in each case, but the original major components, (Neu5Ac)41, (Neu5Ac)53, (Neu5Ac)67 + (Neu5Ac)68, and (Neu5Ac)76 remained intact as the most prominent peaks of quinoxalinone adducts, i.e.(Neu5Ac)40-Q, (Neu5Ac)52-Q, (Neu5Ac)66-Q + (Neu5Ac)67-Q, and (Neu5Ac)75-Q after the DMB reaction. These results showed that the method can be used for the DP determination of free polySia chains with DPs as high as 76. Changes in the polySia content in NCAM have been studied extensively and intensively during the pre- and post-hatching development of central nervous system tissue in vertebrates including chicken and frog (e.g. Ref. 7Sunshine J. Balak K. Rutishauser U. Jacobson M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5986-5990Crossref PubMed Scopus (105) Google Scholar). However, no quantitative information about the developmental stage-dependent changes in the DP of polySia chains is available until now, so that we have applied the DMB/HPLC-FD method to the analysis of polySia chains of NCAM in the samples prepared from embryonic chicken brain at different stages of development. In the present study, polySia-containing glycopeptides were prepared through exhaustive digestion with nonspecific bacterial protease, gel-filtration, and anion-exchange chromatography of the delipidated homogenates of chicken brains at stages E5, E6, E10, E12, E14, E16, E18, and E21 (referred to as 5-, 6-, 10-, 12-, 14-, 16-, 18-, and 21-day after fertilization) by the previously published method and compared with respect to size and distribution of their polySia chains. We analyzed for possible depolymerization of polySia during preparation and purification of samples using model polySia compounds and the HPAEC-PED method. We found that in medium at pH 8.0, used throughout purification, no cleavage of the α2→8 sialosyl linkage was detected during a 3-day incubation at 37 °C. From the results obtained we can conclude that throughout all stages of embryonic development, polysialylated NCAM glycoforms with polySia DP as long as 50 are expressed (Fig. 2, a–d), and although those with a DP > 50 are detectable at some stage (Fig.2 d), they are in much smaller proportion. The distributions of distinct NCAM glycoforms with varying DP polySia chains were estimated at different developmental stages of chicken brain and are shown in Fig. 3. First, NCAM with lower DPs (5 < DP < 10) polySia appeared to be more abundant at early stages of development (E5 and E6) and increased again at late stages (E18 and E21). Second, the highly polymerized form of polySia chains with DP 40, although relatively low over the entire stage of embryonic development, appeared to be most abundant, abounding at around E12 and declining steadily from E14 to the final stage (E21). Third, NCAM glycoforms with a DP greater than 21 increased until the 10th embryonic day and were rather constant between E10 and E16 but decreased significantly from E18. These changes in NCAM glycoforms with varying DPs of polySia chains are considered to be critically important in fine-tuning NCAM-NCAM adhesive interaction-associated cellular events at the defined sites during development, although the complex regulatory mechanism responsible for such fine-tuning remains as one of the most challenging problems confronting us.Figure 3Distribution of polySia chains in different DP ranges are analyzed by the DMB/HPLC-FD method for polySia-glycopeptide samples derived from different stages of embryonic development of chicken brain.View Large Image Figure ViewerDownload (PPT) The chromatographic peaks are well resolved as shown in Fig. 2, but none of these chromatographic patterns are characterized by a damped oscillating fashion with increasing retention time as seen in Fig. 1 for authentic (Neu5Ac)n samples. Instead, regularly protruded peaks are seen in each of chromatograms centering at the retention times near at 50 min (in Fig. 2 a), 44 min (in Fig.2 b), 37 min (in Fig. 2 c), and 46 min (in Fig.2 d). These results are accounted for by the presence of at least two heterogeneous but discrete groups of polySia chains with different DPs. Such unique elution patterns observed in DMB/HPLC-FD analysis were also considered to represent certain kinetic profiles suggesting that particular sialosyl linkages were hydrolyzed at significantly faster rates than α2→8-sialosyl bonds in polySia chains. To further confirm the presence of the discrete groups of polySia chains differing in their average DPs, two pairs of NCAM-derived polySia glycopeptides that were partially resolved by using a MonoQ HR 10/10 column into the high M rfractions at two different developmental stages (E18 and E21) were analyzed by the DMB/HPLC-FD method. As shown in Fig.4of our previous paper (51Cheng M.-C. Lin S.-L. Wu S.-H. Inoue S. Inoue Y. Anal. Biochem. 1998; 260: 154-159Crossref PubMed Scopus (36) Google Scholar), the MonoQ HR 10/10 elution profile for E21 exhibited a minor but definitely partially resolved peak (labeled Q2) eluting with retention times of 38–43 min, just ahead of the major peak (labeled Q1), eluting at 44–52 min. For E18, a similar although less clear trend was seen, and the corresponding major and minor fractions were separately collected and labeled Q1 and Q2, respectively. These two pairs of NCAM-derived polySia-containing glycopeptide fractions were subjected to DMB/HPLC-FD analysis, and the results are shown in Fig. 4,a and b, for E18. and c andd, for E21. The major retarded peaks (Q1) exhibited on a MonoQ HR 10/10 elution profiles of Pronase polySia glycopeptides isolated from E18 and E21 chicken brains are characterized as containing triantennary core N-glycans on which two arms are substituted by oligo/ polySia chains, i.e.(→8Neu5Acα2→)m and (→8Neu5Acα2→)n, andm + n = 50. Elution patterns shown in Fig. 4, a and c, also indicate that only a relatively small portion of the polySia chains has a DP ∼40 and oligo/polySia chains have a high degree of heterogeneity (chain length variability), although a net negative charge is likely almost constant. Data published previously (56Kudo M. Kitajima K. Inoue S. Shiokawa K. Morris H.R. Dell A. Inoue Y. J. Biol. Chem. 1996; 271: 32667-32677Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and unpublished results (S. Inoue) indicate the presence of an arm with an unsubstituted terminal Gal residue on the major triantennary N-glycan bearing oligo/polySia chains. The minor component, Q2, derived from E18 exhibited a unique chromatographic profile (Fig. 4 b), and this fraction can be characterized as containing triantennary glycan chains consisting of two oligo/polySia chains,i.e. (→8Neu5Acα2→)p and (→8Neu5Acα2→)q, p + q = 30, with DPs of 14∼27 predominant. Developmental stage-dependent complex chromatographic patterns observed in this study using the ultrasensitive DMB/HPLC-FD method certainly represent the distinct difference in polysialylation patterns in NCAM from different stages of chicken embryos. During the entire period of embryonic development, a homologous series of NCAM glycoforms categorized by at least two heterogeneous but discrete groups of polySia chains with different DPs are expressed, and their relative abundance is responsible for generating protrusion of a bunch of resolved peaks on chromatograms (Fig. 2). To determine the apparent rate constants of mild acid-catalyzed hydrolysis of ketosidic linkages, appearance of monomeric Neu5Ac and appearance and disappearance of lower oligoNeu5Ac with time were measured in 0.02 m trifluoroacetic acid at 50 °C (some compounds were also measured in 0.1 m trifluoroacetic acid and 80 °C) using the HPAEC-PED method. The rate constants for Neu5Acα2→8Neu5Ac, Neu5Acα2→ 3Galβ1→4Glc and Neu5Acα2→6Galβ1→4Glc were evaluated by a simple first-order rate equation, and those for (→8Neu5Acα2→)10 and (→8Neu5Acα2→)21 were calculated by Equation 1. kapp=−2.303×t−1×[log{(n−1)−(Eq. 1) (n−1)2+(n−2)(N1/Nn−n)}−log(n−2)]Details of the formulation and assumption used are given under "Discussion." The results are summarized in Table I.Table IApparent first-order rate constants, kapp, of mild acid hydrolysis of ketosidic bonds of Neu5Ac ketosidesCompoundReaction conditionsk appmin−1Neu5Acα2→3Galβ1→4Glc50 °C in 0.02 m TFA0.005780 °C in 0.1m TFA0.19Neu5Acα2→6Galβ1→4Glc50 °C in 0.02 m TFA0.003680 °C in 0.1m TFA0.12Neu5Acα2→8Neu5Ac50 °C in 0.02m TFA0.001480 °C in 0.1 mTFA0.033(→8Neu5Acα2→)1050 °C in 0.02m TFA0.0020(→8Neu5Acα2→)2150 °C in 0.02 mTFA0.0023TFA, trifluoroacetic acid. Open table in a new tab TFA, trifluoroacetic acid. In this study developmentally regulated polysialylation of NCAM in chicken brain was analyzed using a newly established ultrasensitive method. This method was validated to be a method to analyze accurate DP values using authentic samples of α2→8-linked polyNeu5Ac with defined DP values up to 76 and to exhibit high sensitivity with detection thresholds of 1.4×10−15 mol (1.4 fmol) per resolved peak. Our results showed that the previously reported estimate of DP of the polySia chains of NCAM (45Livingston B.D. Jacobs J.L. Glick M.C. Troy F.A. J. Biol. Chem. 1988; 263: 9443-9448Abstract Full Text PDF PubMed Google Scholar) is questionable. We also noted that acid lability of polySia chains against hydrolytic cleavage of the inter-residue linkages might not be correctly understood. It is generally believed that polySia chains are substantially labile under mild acidic and even at near neutral conditions, and the lab
KDNα2→3Galβ4Glcβ1Cer [(KDN)GM3] is a major (≈90%) component of total gangliosides found in sperm of rainbow trout ( Oncorhynchus mykiss ) and was shown to be present prominently at the sperm head by immunochemical staining with its specific mAb kdn3G. Liposomes containing (KDN)GM3 adhere specifically to GalNAcβ4Galβ4Glcβ1Cer (Gg3Cer)-coated plastic plates. Interaction between (KDN)GM3 and Gg3Cer was much stronger than that previously observed between Neu5Acα2→3Galβ4Glcβ1Cer and Gg3Cer. (KDN)GM3–Gg3Cer interaction did not require the presence of Ca 2+ and Mg 2+ , but was enhanced in the presence of Mn 2+ . Fresh trout sperm adhered specifically to Gg3Cer-coated plates under physiological conditions, and the binding was inhibited by pretreatment of sperm with mAb kdn3G. The presence of Gg3 or Gg3-related epitope structure in the specific area surrounding the micropyle, through which sperm enter the egg, was confirmed by immunogold labeling under electron microscopy. These findings suggest that initial sperm-egg adhesion during the process of fertilization occurs when sperm adhere to the area surrounding the micropyle through specific interaction between (KDN)GM3 on the sperm head and Gg3 epitope (GalNAcβ4Galβ1→) expressed at a defined region of the egg surface membrane.
