A Unified Rapid PCR Method for Detection of Normal and Expanded Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats in Fragile X Syndrome

We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient’s genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat’s number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.     

Interaction of RNA with transformed glucocorticoid receptor. I. Isolation and purification of the RNA

The glucocorticoid receptor (GR) from mouse AtT-20 pituitary tumor cells, when transformed using a variety of in vitro protocols, yields a DNA-binding RNA-containing 6 S form. In order to better understand the physiological role of RNA interaction with the transformed GR, we have isolated and purified the putative RNA from AtT-20 cells. [3H]Triamcinolone acetonide-labeled cytosolic GR was transformed, using Sephadex G-25 filtration, to yield the RNA-containing 6 S GR. The transformed 6 S GR was separated on DEAE-cellulose into the 4 S GR (eluting at about 100 mM KCl) while its associated RNA eluted at 0.30-0.45 M KCl. The addition of only these RNA fractions to the 4 S GR can reconstitute 6 S GR as shown on 5-20% sucrose gradients. RNA (0.3-0.45 M KCl fractions) was further purified by hydroxylapatite chromatography, and the bound RNA (eluted at approximately 70 mM PO4(-2)) was then loaded onto preparative 5-20% sucrose gradients to separate RNA on the basis of size (sedimentation rate). A uniform class of RNA sedimenting at 4 S was obtained and then adsorbed to oligo(dT)-cellulose columns. The unbound fraction (poly(A-)) was capable of shifting 4 S GR to 6 S. Using these chromatographic procedures about 90% of the cellular RNA, incapable of reconstituting the 6 S GR from the 4 S form, was eliminated. The 4 S GR was covalently cross-linked with the purified RNA (termed PIVB RNA) using formaldehyde. The resulting cross-linked GR X RNA complexes were shown to sediment at the density of ribonucleoprotein (1.38 g/cm3) in CsCl gradients and at the 6 S position in high salt sucrose gradients. The hydrolysis of PIVB RNA with ribonuclease A prevented the formation of high salt-resistant ribonucleoprotein complexes, indicating that the GR may be in close contact with PIVB RNA. Electrophoresis of the PIVB RNA on 5% agarose-formaldehyde-denaturing gels yielded one major band with a molecular size of approximately 75 bases. It thus appears that an endogenous 4 S RNA (PIVB RNA) of about 25 kDa specifically interacts with the monomeric 4 S GR to yield the 6 S GR.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

The next step in biology: A periodic table?

Systems biology is an approach to explain the behaviour of a system in relation to its individual components. Synthetic biology uses key hierarchical and modular concepts of systems biology to engineer novel biological systems. In my opinion the next step in biology is to use molecule-to-phenotype data using these approaches and integrate them in the form a periodic table. A periodic table in biology would provide chassis to classify, systematize and compare diversity of component properties vis-a-vis system behaviour. Using periodic table it could be possible to compute higher-level interactions from component properties. This paper examines the concept of building a bio-periodic table using protein fold as the fundamental unit.     

A new species of water mite <Emphasis Type="Italic">Atractides</Emphasis> (<Emphasis Type="Italic">Atractides</Emphasis>) <Emphasis Type="Italic">turcicus</Emphasis> sp. n. (Acari: Hydrachnidia: Hygrobatidae) from Turkey

In this study, the structural characteristics, unique features, various organ measurements of males and females of the water mite Atractides (Atractides) turcicus sp. n. from Turkey are described. In addition, the study compares their characteristics with related species.     

Getting to the heart of the matter: CCN2 plays a role in cardiomyocyte hypertrophy

Connective tissue growth factor (CTGF/CCN2) is overexpressed in diabetes. Diabetic rats possess myocardial and cardiomyocyte hypertrophy. In a recent report, Wang and colleagues (Am J Physiol Cell Physiol. 2009 Jul 22. [Epub ahead of print]) show that CCN2 directly mediates cardiomyocyte hypertrophy as well as that induced by high glucose and fatty acid. CCN2 acted via the TrkA receptor. These data are the subject of this commentary, and emphasize that CCN2 may be an excellent target for therapy in diabetes.