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.     

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.     

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.     

The ability of the biological control agent Bacillus subtilis, strain BB, to colonise vegetable brassicas endophytically following seed inoculation

The ability of Bacillus subtilis, strain BB, to colonise cabbage seedlings endophytically was examined following seed inoculation. Strain BB was recovered from different plant parts including leaves (cotyledons), stem (hypocotyl) and roots. While high bacterial populations persisted in the roots and lower stem, they were lower in the upper stem and leaves through time. In addition to cabbage, strain BB colonised endophytically the roots of 5 other vegetable brassicas. Fatty acid methyl ester (FAME) and PCR fingerprinting analysis confirmed the reliability of the detection method. Studies conducted with transmission electron microscope (TEM) showed that BB mainly colonised intercellular spaces of cortical tissues including intercellular spaces close to the conducting elements of roots and stem of cabbage seedlings. Gold labelling was specifically associated with BB and the fibrillar material filling the intercellular spaces where bacterial cells were found.     

Mate detection success of maleClethrionomys rufocanus in relation to the spatial distribution of sexually receptive females

Summary Mate detection success of male grey-sided voles,Clethrionomys rufocanus, in relation to the spatial distribution of sexually receptive females was studied in an experimental island population. The spatiotemporal distribution of receptive females was controlled by containing females in small, mobile wire-mesh cages, whereas the response by free-ranging males was monitored by means of radiotelemetry. Males were on average more successful in finding oestrous females when females were spatially clumped than when females were spatially overdispersed. In addition, the variance (CV) in male mate detecting success was highest when females had an overdispersed spatial distribution. These results are consistent with predictions from a theoretical model (Ims, 1988b) analysing the effect of mate distribution on male mating success, and with empirical results on prey detection success of predators searching for prey.     

Genome‐wide detection of CNVs associated with beak deformity in chickens using high‐density 600K SNP arrays

Beak deformity (crossed beaks) is found in several indigenous chicken breeds including Beijing‐You studied here. Birds with deformed beaks have reduced feed intake and poor production performance. Recently, copy number variation (CNV) has been examined in many species and is recognized as a source of genetic variation, especially for disease phenotypes. In this study, to unravel the genetic mechanisms underlying beak deformity, we performed genome‐wide CNV detection using Affymetrix chicken high‐density 600K data on 48 deformed‐beak and 48 normal birds using penncnv . As a result, two and eight CNV regions (CNVRs) covering 0.32 and 2.45 Mb respectively on autosomes were identified in deformed‐beak and normal birds respectively. Further RT‐qPCR studies validated nine of the 10 CNVRs. The ratios of six CNVRs were significantly different between deformed‐beak and normal birds (P < 0.01). Within these six regions, three and 21 known genes were identified in deformed‐beak and normal birds respectively. Bioinformatics analysis showed that these genes were enriched in six GO terms and one KEGG pathway. Five candidate genes in the CNVRs were further validated using RT‐qPCR. The expression of LRIG2 (leucine rich repeats and immunoglobulin like domains 2) was lower in birds with deformed beaks (P < 0.01). Therefore, the LRIG2 gene could be considered a key factor in view of its known functions and its potential roles in beak deformity. Overall, our results will be helpful for future investigations of the genomic structural variations underlying beak deformity in chickens.     

Separation of enantiomeric amines and acids using chiral ion-pair chromatography on porous graphitic carbon

The application of porous graphitic carbon as adsorbing phase for direct separation of enantiomeric acids and amines using chiral ion-pair chromatography is described. The enantiomeric amines were separated as diastereomeric ion pairs with N-benzyloxycarbonylglycyl-L -proline, N-benzyloxycarbonylglycylglycyl-L -proline, or captopril as the chiral counterion. High enantioselectivities were obtained for amines having a hydrogen bonding function in the vicinity of the asymmetrical carbon atom. Quinine was the chiral counterion used to separate the enantiomeric acids. The strongly UV-absorbing quinine improved detection of solutes having low UV-absorbing properties, e.g., (R,S)-2-chloropropionic acid, by “indirect detection.” Retention and stereoselectivity of enanticmeric acids were regulated by the quinine concentration and by the addition of carboxylic acids as well as polar modifiers, e.g., methanol and 2-propanol, to the mobile phase. © 1992 Wiley-Liss, Inc.     

Possible Phytoplasma Disease in Papaya (Carica papaya L.) from Baja California Sur: Diagnosis by Scanning Electron Microscopy

The symptoms of possible phytoplasma infection in introduced and local varieties of papaya were first noted in the Mexican state of Baja California Sur (BCS) during field surveys in 2002–2003. Phytoplasma structures were observed using scanning electron microscopy (SEM) in phloem sieve elements in diseased papaya plants, but not in healthy plants. They were rounded structures 400–1600 nm in diameter. This is the first report of the possible association of phytoplasmas with diseased papaya plants in BCS. The use of SEM for the primary detection of disease aetiology is discussed.     

Cadmium-containing granules in kidney tissue of the Atlantic white-sided dolphin (Lagenorhyncusacutus) off the Faroe Islands

Top predators from the northern sub-polar and polar areas exhibit high cadmium concentrations in their tissues. In the aim to reveal possible adverse effects, samples of five Atlantic white-sided dolphins Lagenorhyncusacutus have been collected on the occasion of the drive fishery in the Faroe Islands, for ultrastructural investigations and energy dispersive X-ray microanalyses. Cadmium concentrations were less than the limit of detection in both immature individuals and ranged from 22.7 to 31.1 μg g^?1 wet weight in the mature individuals. Two individuals with the highest cadmium concentrations exhibited electron dense mineral concretions in the basal membranes of the proximal tubules. They are spherocrystals made up of numerous strata mineral deposit of calcium and phosphorus together with cadmium. Cadmium has been detected with a molar ratio of Ca:Cd of 10:1 in the middle of these concretions. To our knowledge, this is the first report of such granules in a wild vertebrate. The role of these granules in the detoxification of the metal and the possible pathological effects are considered.     

Progeny testing for recessive genes: procedures and interpretations

Summary Methods for calculating the probability of detecting a carrier of a recessive gene by utilizing matings among related individuals are presented for single and litter bearing species. The confidence level for detection of heterozygosity depends upon: (1) the genetic relationship between mates, (2) the number of mates per male and the number of offspring per mate, (3) whether an estimate of recessive gene frequency before selection is available and (4) the magnitude of that frequency. Methods of computing probability of heterozygosity vs homozygosity utilizing Bayes theorem also are presented. In the conventional progeny test method, a sire initially is assumed heterozygous before calculations are made, but no prior information concerning his probable genotype is utilized. In the method using Bayes theorem, prior sources of information from relatives or from estimates of population allele frequency are utilized. This method gives the exact probability that a sire is not a carrier, given prior information and that he produces all normal offspring. These methods could be used in any sexually reproducing species to identify not only detrimental genes but beneficial genes as well.