Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Characterization of components released by alkali disruption of simian virus 40.

Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were host cell histones, but SV40-specific proteins VP3 and VP2 were also present. The 7S protein particles were almost exclusively VP1 and, in negatively stained samples, resembled the capsomer structures of intact virions.     

Physical and Genetic Characterization of Deletion Mutants of Simian Virus 40 Constructed In Vitro

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.     

Potential of alfalfa as an alternative molt induction diet for laying hens: egg quality and consumer acceptability

Dietary molt induction to initiate additional egg laying cycles in commercial laying hen flocks is a wide spread practice in the United States. Feed deprivation is the most commonly used method but this practice has generated several concerns which has lead to research for viable alternative approaches. From a management standpoint a single ingredient molting diet consisting of high fiber-low energy represents an easily adaptable diet for large laying hen production units. Alfalfa meal is readily available in most commercial locations and possesses many of the desirable properties of an ideal laying hen molt diet. In the current study hens at a commercial laying facility were molted by both alfalfa and feed deprivation. After the hens had reentered post-molt commercial egg production, eggs were examined for egg quality performance. Egg shell strength, albumen height, yolk height, weight, length, and yolk color were all tested using various mechanical techniques. The eggs were also sampled for testing by consumer sensory panels that assessed the desirability of the eggs' color and flavor/texture. Eggs laid by hens molted by alfalfa had a significantly lower (p<0.05) "a*" level of colorimetry. Eggs laid by hens molted with alfalfa also exhibited significantly higher (p<0.05) egg weights and length. In the consumer sensory test, there was no significant difference (p>0.05) in color or flavor/texture scores in eggs from either feed deprived or alfalfa molted hens. The consumer sensory and mechanical quality attributes indicates that alfalfa shows promise as an alternative molt induction diet by providing a single diet option for extending egg production into a second egg laying cycle.     

Kinetic models for association of 2,3,7,8-tetrachlorodibenzo-p-dioxin with the Ah receptor

Saturation binding studies of the interaction between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the Ah receptor obtained from the hepatic cytosol of Wistar rats have been carried out. The conventional Scatchard analysis for determination of the equilibrium constant for ligand-receptor binding has been shown to be inappropriate due to thermal inactivation of the unoccupied receptor. Simulation models of the receptor-ligand binding kinetics which take into account receptor degradation have been developed and the results are consistent with two alternative kinetic models. In Model 1, reversible 2,3,7,8-TCDD-receptor binding occurs in parallel with inactivation of the unbound receptor; analysis of the observed data using this model suggests that the previously determined equilibrium constants (Kass) for association of the ligand with the receptor are orders of magnitude too low and the total initial receptor concentrations are somewhat underestimated. In Model 2, the unbound receptor is converted unimolecularly to an activated state which then undergoes competitive degradation or entrapment by ligand. Experiments have been carried out over the temperature range 4-37 degrees C, enabling activation parameters to be obtained. According to Scheme 1, the activation enthalpies for association of receptor with ligand and for thermal inactivation of the unoccupied receptor are high, and numerically almost identical (delta H++ ca 125 kJ mol-1). These reactions are strongly entropically driven and this is consistent with association being accompanied by a conformational change in the receptor protein, and the previously postulated binding of the ligand to a hydrophobic pocket. According to Scheme 2, there is only one enthalpy of activation because both inactivation and entrapment by 2,3,7,8-TCDD are fast processes which follow the same slow activation step. On the basis of this latter model, a 10(-9) M concentration of 2,3,7,8-TCDD is sufficient to trap roughly two-thirds of the activated receptors.     

Avian biodiversity in the coastal wetlands of the Okahukura Peninsula

The Tapora Landcare Group, operating on the Okahukura Peninsula, has the long-term goal of making this region predator fenced. The aim of this study was to obtain information on the current status of avian biodiversity and the bird community across the band of coastal wetlands on the Okahukura Peninsula. Bird counts were conducted and playback lures used to detect three cryptic wetland species: fernbirds (Bowdleria punctata); spotless crakes (Porzana tabuensis); and banded rails (Gallirallus philippensis). Fernbirds and banded rails were detected at seven of the eight wetland sites sampled whereas spotless crakes were detected at two sites. The native species with the highest relative abundance across the eight sites were silvereyes (Zosterops lateralis) and South Island pied oystercatchers (Haematopus finschi). Changes in avian biodiversity over time in the region can now be monitored, and comprehensive long-term data on the status of avian biodiversity over time obtained.     

Effect of oxygen concentrations on sodium iodide symporter expression and iodide uptake and hCG expression in human choriocarcinoma BeWo cells

Normal human fetal development requires an adequate supply of thyroid hormone from conception. Until about 16 wk gestation this is supplied entirely by placental transfer of maternal hormone. Subsequently, the fetal thyroid synthesizes thyroid hormones, requiring a supply of maternal iodide. Trophoblast iodide transfer is mediated by the apical sodium iodide symporter (NIS). Placental oxygen levels are low in early pregnancy (~1%), rising with placental vascularisation to a plateau of ~8% at about 16 wk. Although the impact of these changing oxygen levels on placental implantation is well recognized, effects on trophoblast materno-fetal exchange are less understood. We investigated expression of the NIS regulator hCG, NIS mRNA expression, and I(125) uptake in choriocarcinoma BeWo cells (a model of the trophoblast) cultured in 1 and 8% oxygen and in room air (21% oxygen). Expression of NIS and hCG mRNA and protein was low at 1% oxygen but rose significantly at 8 and at 21%. This was reflected in significant increases in I(125) uptake. Desferrioxamine, an iron chelator and hypoxia mimic, decreased NIS and hCG expression and I(125) uptake in BeWo cells. NIS expression and I(125) uptake in cells grown at 1% oxygen were not increased by addition of hCG (2,500 IU/l). We infer that placental NIS mRNA and protein expression are regulated by oxygen, rising with vascularization of the placenta in the late first trimester, a time when fetal iodide requirements are increasing.     

SiO2/SnO2/Sb2O5 microporous ceramic material for immobilization of Meldola's blue: application as an electrochemical sensor for NADH

The mixed oxide SiO(2)/SnO(2), containing 25 wt% of SnO(2), determined by X-ray fluorescence, was prepared by the sol-gel method and the porous matrix obtained was then grafted with Sb (V), resulting the solid designated as (SiSnSb). XPS indicated 0.7% of Sb atoms on the surface. Sb grafted on the surface contains Br?nsted acid centers (SbOH groups) that can immobilize Meldola's blue (MB(+)) cationic dye onto the surface by an ion exchange reaction, resulting the solid designated as (SiSnSb/MB). In the present case a surface concentration of MB(+)=2.5×10(-11) mol cm(2) on the surface was obtained. A homogeneous mixture of the SiSnSb/MB with ultra pure graphite (99.99%) was pressed in disk format and used to fabricate a working electrode that displayed an excellent specific electrocatalytic response to NADH oxidation, with a formal potential of -0.05 V at pH 7.3. The electrochemical properties of the resulting electrode were investigated thoroughly with cyclic voltammetric and chronoamperometry techniques. The proposed sensor showed a good linear response range for NADH concentrations between 8×10(-5) and 9.0×10(-4) mol L(-1), with a detection limit of 1.5×10(-7) mol L(-1). The presence of dopamine and ascorbic acid did not show any interference in the detection of NADH on this modified electrode surface.     

赖氨酰氧化酶与人类疾病

赖氨酰氧化酶(lysyl oxidases,LOXs)是一种能够催化细胞外基质蛋白(如胶原和弹性蛋白)交叉连接的酶类,这一功能使其在组织的稳定、重塑和伤口愈合中发挥重要作用.随着研究的不断深入,LOXs在细胞增殖、细胞趋化以及肿瘤发生等过程中也彰显出十分关键的作用.研究发现,一些诸如结缔组织病、剥脱综合症、铜代谢障碍性疾病及盆腔器官脱垂和骨疾等疾病的发生与LOXs有很大关系.综述了LOXs的生物合成、结构特点、多功能性以及与人类疾病的关系.