Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic

Conversion of thyroxine (T-4) to 3,5,3'-triiodothyronine is an essential first step in controlling thyroid hormone action. Type I deiodinase (DI) can catalyze the conversion to produce the bulk of serum 3,5,3'-triiodothyronine. Acting as a mimic of DI, a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against T4 into selenocysteines, can catalyze the deiodination of T4 with dithiothreitol (DTT) as cosubstrate. The mimic enzyme Se-4C5 exhibited a much greater deiodinase activity than model compound ebselen and another selenium-containing antibody Se-Hp4 against GSH. The coupling of selenocysteine with the combining pocket of antibody 4C5 endowed Se-4C5 with enzymatic activity. To probe the catalytic mechanism of the catalytic antibody, detailed kinetic studies were carried out in this paper. Investigations into the deiodinative reaction revealed the relationship between the initial velocity and substrate concentration. The characteristic parallel Dalziel plots demonstrated that Se-4C5-catalyzed reaction mechanism was ping-pong one, involving at least one covalent enzyme intermediate. The kinetic properties of the catalytic antibody were similar to those of DI, with K-m values for T-4 and DTT of approximately 0.8 muM and 1.8 muM, respectively, and a V-m value of 270 pmol per mg of protein per min. The activity could be sensitively inhibited by 6-propyl-2-thiouracil (PTU) with a K-i value of similar to 120 muM at 2.0 muM T-4 concentration. The PTU inhibition was progressively alleviated with the increasing concentration of added DTT, revealing that PTU was a competitive inhibitor for DTT.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

The search for the IFN-gamma receptor in fish: Functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFN gamma R1 and IFN gamma R2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFN gamma Rs in fish. They are widely expressed in tissues, with IFN gamma R1 typically more highly expressed than IFN gamma R2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1 beta down regulating IFN gamma R1 expression but up regulating IFN gamma R2 expression. Overexpression of the two receptor chains in RTG-2 cells revealed that the level of IFN gamma R2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gamma IP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFN gamma R may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction. (c) 2009 Elsevier Ltd. All rights reserved.

Identification of an additional two-cysteine containing type I interferon in rainbow trout Oncorhynchus mykiss provides evidence of a major gene duplication event within this gene family in teleosts

Multiple type I interferons (IFNs) have recently been identified in salmonids, containing two or four conserved cysteines. In this work, a novel two-cysteine containing (2C) IFN gene was identified in rainbow trout. This novel trout IFN gene (termed IFN5) formed a phylogenetic group that is distinct from the other three salmonid IFN groups sequenced to date and had a close evolutionary relationship with IFNs from advanced fish species. Our data demonstrate that two subgroups are apparent within each of the 2C and 4C type I IFNs, an evolutionary outcome possibly due to two rounds of genome duplication events that have occurred within teleosts. We have examined gene expression of the trout 2C type I IFN in cultured cells following stimulation with lipopolysaccharide, phytohaemagglutinin, polyI:C or recombinant IFN, or after transfection with polyI:C. The kinetics of gene expression was also studied after viral infection. Analysis of the regulatory elements in the IFN promoter region predicted several binding sites for key transcription factors that potentially play an important role in mediating IFN5 gene expression.

Phylogeny and speciation of the eastern Asian cyprinid genus Sarcocheilichthys

The genus Sarcocheilichthys is a group of small cyprinid fishes comprising 10 species/sub-species widely distributed in East Asia, which represents a valuable model for understanding the speciation of freshwater fishes in East Asia. In the present study, the molecular phylogenetic relationship of the genus Sarcocheilichthys was investigated using a 1140 bp section of the mitochondrial cytochrome b gene. Two different tree-building methods, maximum parsimony (MP) and Bayesian methods, yielded trees with almost the same topology, yielding high bootstrap values or posterior probabilities. The results showed that the genus Sarcocheilichthys consists of two large clades, clades I and II. Clade I contains Sarcocheilichthys lacustris, Sarcocheilichthys sinensis and Sarcocheilichthys parvus, with S. parvus at a basal position. In clade II, Sarcocheilichthys variegatus microoculus is at a basal position; samples of the widespread species, Sarcocheilichthys nigripinnis, form a large subclade containing another valid species Sarcocheilichthys czerskii. Sarcocheilichthys kiangsiensis is retained at an intermediate position. Since S. czerskii is a valid species in the S. nigripinnis clade, remaining samples of S. nigripinnis form a paraphyly. This speciation process is attributed to geographical isolation and special environmental conditions experienced by S. czerskii and stable environments experienced by the other S. nigripinnis populations. This type of speciation process was suggested to be very common. Samples of Sarcocheilichthys sinensis sinensis and Sarcocheilichthys sinensis fukiensis that did not form their own monophyletic groups suggest an early stage of speciation and support their sub-species status. Molecular clock analysis indicates that the two major lineages of the genus Sarcocheilichthys, clades I and II diverged c. 8.89 million years ago (mya). Sarcocheilichthys v. microoculus from Japan probably diverged 4.78 mya from the Chinese group. The northern-southern clades of S. nigripinnis began to diverge c. 2.12 mya, while one lineage of S. nigripinnis evolved into a new species, S. czerski, c. 0.34 mya. (C) 2008 The Authors Journal compilation (C) 2008 The Fisheries Society of the British Isles.

Observation of quantum-confined Stark shifts in SiGe/Si type-I multiple quantum wells

Quantum-confined Stark shifts in SiGe/Si type-I multiple quantum wells are suggested by the bias dependence of the photocurrent spectra of p-i-n photodiodes. Both Stark redshift and blueshift have been observed for the same sample in the different ranges of electric fields applied to the quantum wells. The turnaround point corresponds to a certain electric field (named "critical" field). This phenomenon was generally predicted by Austin in 1985 [Phys. Rev. B 31, 5569 (1985)] and calculated in detail for SiGe quantum structure by Kim recently [Thin Solid Films 321, 215 (1998)]. The critical electric field obtained from the photocurrent spectra is in reasonable agreement with the theoretical prediction. (C) 2000 American Institute of Physics. [S0021-8979(00)03711-7].

Structural analysis of PEKEKK(T/I) using WAXD, imaging plates and SAXS

Structures and crystal form transition of the novel aryl ether ketone polymer containing meta-phenylene linkage: PEKEKK(T/I) were investigated by wide angle X-ray diffraction (WAXD), imaging plates (IPs) and small angle X-ray scattering (SAXS). The energy of activation of the decomposition reaction and degree of crystallinity of PEKEKK(T/I) were determined by WAXD and thermo-gravimetric analysis (TGA), respectively. Results obtained from WAXD and IPs show that crystal forms I and II coexist in the PEKEKK(T/I) samples isothermally cold crystallized in the temperature range from 180degreesC to 240degreesC and only form I occurs in PEKEKK(T/I) samples isothermally cold crystallized at 270degreesC. The radius of gyration (Rg), thickness of microregions with electron-density fluctuations (E) and distribution of particle sizes were investigated by SAXS.

A selenium-containing catalytic antibody with type I deiodinase activity

Acting as a mimic of type I deiodinase (DI), a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against thyroxine (T-4) into selenocysteines, can catalyze the deiodination of T-4 to 3,5,3'-triiodothyronine (T-3) with dithiothreitol (DTT) as cosubstrate. Investigations into the deiodinative reaction by Se-4C5 revealed the relationship between the initial velocity and substrate concentration was subjected to Michaelis-Menten equation and the reaction mechanism was ping-pong one. The kinetic properties of the catalytic antibody were a little similar to those of DI, with K-m values for T-4 and DTT of approximately 0.8 muM and 1.8 mM, respectively, and V-m value of 270 pmol per mg protein per min. The activity could be sensitively inhibited by PTU with a K-i value of approximately 120 muM at 2.0 muM of T-4 concentration, revealing that PTU was a competitive inhibitor for DTT, (C) 2001 Academic Press.

Theoretical research on noncollinear match conditions of the type I optical parametric process

Broad bandwidth group match conditions are reported for a noncollinear type I optical parametric process. The theoretical calculations corresponding to two special situations in practice were made, respectively, which are in accordance with the published experimental results. Furthermore, we provide a method to not only achieve maximal parametric bandwidth output but also match the group velocities between three waves. (c) 2006 Optical Society of America.

Mitochondrial DNA variation in cattle of South China: origin and introgression

Ten restriction endonucleases were used to investigate the mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) of 11 native cattle breeds and one cultivated cattle breed in South China. Twenty-three restriction morphs were detected, which can be sorted into five haplotypes, A phylogenetic tree of the haplotypes was constructed by using the 'upgma' method. Our study showed that haplotype I and II are identical to the zebu (Bos indicus) and taurine (Bos taurus) haplotypes, respectively. Zebu and taurine were the two major origins of cattle populations in South China, and the zebu probably had more influence on the native cattle population than taurine did. Haplotype III is identical to haplotype I of yak (Bos grunniens), which was only detected in the Diqing cattle breed. Haplotype IV was detected for the first time. This haplotype, found only in Dehong cattle, might be from an independent domestication event, probably from another Bos indicus population. Divergence of haplotypes I and IV occurred about 268,000-535 000 years ago, much earlier than the 10,000-year history of cattle husbandry. Our results also suggest a secondary introgression of mtDNA from yak to Diqing cattle.

Spatiotemporal heterogeneity of plankton communities in Lake Donghu, China, as revealed by PCR-denaturing gradient gel electrophoresis and its relation to biotic and abiotic factors

The 16S and 18S rRNA genes of planktonic organisms derived from five stations with nutrient gradients in Lake Donghu, China, were studied by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, and the relationships between the genetic diversity of the plankton community and biotic/abiotic factors are discussed. The concentrations of total nitrogen (TN), total phosphorus (TP), NH4-N and As were found to be significantly related (P < 0.05) to morphological composition of the plankton community. Both chemical and morphological analyses suggested that temporal heterogeneity was comparatively higher than spatial heterogeneity in Lake Donghu. Although the morphological composition was not identical to the DGGE fingerprints in characterizing habitat similarity, the two strongest eutrophic stations (I and II) were always initially grouped into one cluster. Canonical correspondence analysis suggested that the factors strongly correlated with the first two ordination axes were seasonally different. The concentrations of TN and TP and the densities of rotifers and crustaceans were generally the main factors related to the DGGE patterns of the plankton communities. The study suggested that genetic diversity as depicted by metagenomic techniques (such as PCR-DGGE fingerprinting) is a promising tool for ecological study of plankton communities and that such techniques are likely to play an increasingly important role in assessing the environmental conditions of aquatic habitats.

Genetic diversity of plankton community as depicted by PCR-DGGE fingerprinting and its relation to morphological composition and environmental factors in lake donghu

To collect information about the genetic diversity of the plankton community and to study how plankton respond to environmental conditions, plankton samples were collected from five stations representing different trophic levels in a shallow, eutrophic lake (Lake Donghu), and investigated by PCR-DGGE fingerprinting. A total of 100 bands (61 of 16S rDNA bands and 39 of 18S rDNA bands) were detected. The DGGE bands unique to any single station accounted for 38% of the total bands, whereas common bands detected at all five stations accounted for only 11%. Using UPGMA clustering and MDS ordination of DGGE fingerprints, stations I and II were found to initially group together into one cluster, which was later joined by station V. Stations III and IV were isolated into two separate groups of one station each. Some differences in grouping relationships were found when analysis was completed on the basis of chemical characteristics and morphological composition, with zooplankton composition showing the greatest variability. However, the most similar stations (I and II) were always initially grouped into one cluster. Moreover, stations that exhibited the same or similar trophic level (stations III and IV), but different concentrations of heavy metals, were further differentiated by the DGGE method. Results of the present study indicated that PCR-DGGE fingerprinting was more sensitive than the traditional methods, as other studies suggested. Additionally, PCR-DGGE appears to be more appropriate for diversity characterization of the plankton community, as it is more canonical, systematic, and effective. Most importantly, fingerprinting results are more convenient for the comparative analyses between different studies. Therefore, the use of the described fingerprinting analysis may provide an operable and sensitive biomonitoring approach to identify critical, and potentially negative, stress within an aquatic ecosystem.

Sedimentation rates, nitrogen and phosphorus retentions in the largest urban Lake Donghu, China

Sediment core samples were collected in the largest urban Lake Donghu (Stations I and II) in China, and the activities of Pb-210, Ra-226 and Cs-137 were measured by gamma-ray spectrometry. The sedimentation rates, calculated by 210Pb constant rate of supply (CRS) model, ranged from 0.11 to 0.65 (average 0.39) cm(.)y(-1) at Station I, and from 0.21 to 0.78 (average 0.46) cm(.)y(-1) at Station II. Sedimentation rate calculated by Cs-137 as a time marker was 0.55 cm(.)y(-1) at Station II. Based on the average sedimentation rate, we obtained 769 and 147 t(.)y(-1) for nitrogen and phosphorus retentions in Lake Donghu sediments, respectively.

Structure of the phytoplankton community and its relationship to water quality in Donghu Lake, Wuhan, China

The phytoplankton community structure, in terms of species composition, total standing crop, and abundance of the dominant algal species, at four stations in Donghu Lake, Wuhan, China, was investigated monthly from January 1994 to December 1996. A total of 260 taxa was observed, of which Chlorophyta (106 taxa) contributed the highest portion of the total number of taxa, followed by Bacillariophyta (82 taxa) and Cyanophyta (32 taxa). The total standing crop measured by means of chlorophyll a content, cell density, and cell biovolume, as well as the abundance of the dominant species, declined in the order of Station I to Station IV. Seasonal changes of the standing crop varied greatly among the four stations. Although the cell density at the four stations showed a single peak within a year, the peak density varied from July to November, dependent on the sampling year and the station. For chlorophyll a content and cell biovolume, multiple peaks were observed at Stations I and II, but a single peak was found at Stations III and IV. The phytoplankton community structure indicated that the trophic status was the highest at Station I (most eutrophic), followed by Station II; Stations III and IV were the least trophic areas. The long-term changes in phytoplankton community structure further suggested that changes in phytoplankton community structure were correlated with water quality, and eutrophication of Donghu Lake had been aggravated since the 1950s.

The role of plants in channel-dyke and field irrigation systems for domestic wastewater treatment in an integrated eco-engineering system

Eight kinds of plants were tested in channel-dyke and field irrigation systems. The removal rates of TP, phosphate, TN, ammonia, CODcr and BOD, in the channel-dyke system with napiergrass (Pennisetum purpurem Schumach, x Pennisetum alopecuroides (L.) Spreng American) were 83.2, 82.3, 76.3, 96.2, 73.5 and 85.8%, respectively. The field irrigation systems with rice I-yuanyou No.1(88-132) (Oryza sativa L.) and rice II- suakoko8 (Oryza glaberrima) had high efficiency for N removal; the removal rate were 84.7 and 84.3%, respectively. The mass balance data revealed that napiergrass, rice I and II were the most important nutrient sinks, assimilating more than 50% of TP and TN. Plant uptake of N and P as percentage of total removal from wastewater correlated with biomass yield of and planting mode. (C) 2000 Elsevier Science B.V. All rights reserved.