A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites

Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for β-glucosidase (GBA) and the β-polypeptide 1 of the Na⁺, K⁺-transporting ATPase (ATP1B1). Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097). The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88 CM. This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene. In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4pl2-pl3. The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E. coli pilus antigens. One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization.     

In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes

In vitro as well as in vivo evaluation of the reactivating efficacy of various oximes against nerve agent-inhibited acetylcholinesterase has been usually done with the help of animal experiments. Nevertheless, previously published data indicate that the reactivation potency of oximes may be different in human and animal species, which may hamper the extrapolation of animal data to human data. Therefore, to better evaluate the efficacy of various oximes (pralidoxime, obidoxime, HI-6, K033) to reactivate brain acetylcholinesterase inhibited by sarin by in vitro methods, human, rat and pig brain acetylcholinesterase were used to calculate kinetic parameters for the reactivation. Our results show differences among the species, depending on the type of oxime, and indicate that data from animal experiments needs to be carefully evaluated before extrapolation to humans.     

Prenatal diagnosis of Pallister-Killian syndrome and literature review

Pallister-Killian syndrome (PKS) is a rare sporadic genetic disorder usually caused by mosaicism of an extra isochromosome of 12p (i(12p)). This retrospective study analysed the prenatal ultrasound manifestations and molecular and cytogenetic results of five PKS foetuses. Samples of amniotic fluid and/or cord blood, skin biopsy and placenta were collected. Conventional karyotyping and single nucleotide polymorphism array (SNP array) were performed on all the amniotic fluid or cord blood samples. Copy number variants sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were also used for the validation for one foetus. All the five foetuses were from pregnancies with advanced parental age. Two foetuses involved structural abnormalities and one foetus had only soft markers, all of which included increased nuchal translucency. The rest two foetuses had normal ultrasounds in the second trimester, which has rarely been reported before. The karyotype revealed typical i(12p) in four cases and a small supernumerary marker chromosome consisting of 12p and 20p in the remaining one case. The proportion of cells with i(12p) ranged from 0 to 100% in cultural cells, while SNP array results suggested 2−4 copies of 12p. For one foetus, metaphase FISH showed normal results, but the interphase FISH suggested cell lines with two, three and four copies of 12p in the amniotic fluid. Advanced parental age may be an important risk factor for PKS, and there were no typical ultrasound manifestations related to PKS. A combination of karyotype analysis and molecular diagnosis is an effective method for the diagnosis of PKS.     

The impact of hybrids between genetically modified crop plants and their related species: introgression and weediness

Assessing the impact of hybrids between transgenic plants and nontarget wild species involves answering several questions such as: (i) what are the hybridization and introgression rates; (ii) what is the behaviour of a transgene in a wild population; and (iii) what will be the consequences of the expression of a transgene in a wild population? These issues are discussed using results from experiments on oilseed rape and wild related Brassiceae. Evidence is given of large variations in the estimates of cross-fertilization probabilities. The first stage of introgression into wild populations is demonstrated to occur spontaneously through back-crossing. Population analysis may also be valuable to detect traces of past introgression. Data from the literature on weed biology, and especially herbicide resistance, are used to illustrate the behaviour of a new gene in weed populations. The need for computer models simulating the introgression process is stressed.     

The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter

The influence of low temperature on the operation of the xanthophyll cycle and energy dissipation activity, as ascertained through measurements of chlorophyll fluorescence, was examined in two broad-leaved evergreen species, Vinca minor L. and Euonymus kiautschovicus Loessner. In leaves examined under laboratory conditions, energy dissipation activity developed more slowly at lower leaf temperatures, but the final, steady-state level of such activity was greater at lower temperatures where the rate of energy utilization (through photosynthetic electron transport) was much lower. The rate at which energy dissipation activity increased was similar to that of the de-epoxidation of violaxanthin to antheraxanthin and zea-xanthin at different temperatures. However, leaves in the field examined prior to sunrise on mornings following cold days and nights exhibited a retention of antheraxanthin and zeaxanthin that was associated with sustained decreases in photosystem II efficiency. We therefore suggest that this phenomenon of ‘photoinhibition’ in response to light and cold temperatures during the winter results from sustained photoprotective thermal energy dissipation associated with the xanthophyll cycle. Such retention of the de-epoxidized components of the xanthophyll cycle responded to day-to-day changes in temperature, being greatest on the coldest mornings (when photoprotective energy dissipation might be most required) and less on warmer mornings when photosynthesis could presumably proceed at higher rates.     

COMPARISON OF VERTICAL AERIAL PHOTOGRAPHIC AND GROUND CENSUSES OF STELLER SEA LIONS AT AÑO NUEVO ISLAND, JULY 1990-1993

Counts of Steller sea lion ( Eumetopias jubatus ) pups and non-pups (adults and juveniles) from aerial photographs of rookeries at Año Nuevo Island between 1990 and 1993 were significantly higher than those made on the ground. Based on regression of natural logs of photographic counts versus year, the number of pups declined at a rate of −0.099yr while non-pup numbers declined at −0.315/yr. Examination of ground count data for the same period revealed a significant decline in non-pups (−0.139/yr), but no trend was detected in the ground counts of pups. The regression coefficients from photographic and ground counts of non-pups did not differ significantly. Power analyses using the program TRENDS indicated that detectable rates of change in abundance from four annual surveys were much lower for counts of pups than counts of non-pups where sampling precision was based on fits to linear models.     

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.     

Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Biological characterization of the lytic cycle of actinophage φA7 in Streptomyces antibioticus

Some basic parameters of the lytic development of phage phi A7 in Streptomyces antibioticus are described. One-step growth experiments demonstrated that at 28 degrees C phi A7 has a latent period of about 60 min and an exponential growth period of about 35 min. The average burst size ranged from 70-100 plaque forming units per infected cell. At the same temperature 50% of the virions were adsorbed to germ tubes of S. antibioticus in about 10 min. This corresponds to an adsorption constant of 6.5 x 10(-10) ml/min. The phage was unable to adsorb the host at other stages of the life cycle (spores or mycelium). Divalent cations are not required for phi A7 stability but Ca2+ proved to be essential for adsorption and also for a later stage of the vegetative development of the phage.