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.     

Group-selective reagent modification of the sodium- and chloride-coupled glycine transporter under native and reconstituted conditions.

Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the Vmax for glycine transport and to a lesser extent act on the apparent Km. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.     

Views,landmarks, and routes: how do desert ants negotiate an obstacle course?

The Australian desert ant Melophorus bagoti often follows stereotypical routes through a cluttered landscape containing both distant panoramic views and obstacles (plants) to navigate around. We created an artificial obstacle course for the ants between a feeder and their nest. Landmarks comprised natural objects in the landscape such as logs, branches, and tussocks. Many ants travelled stereotypical routes home through the obstacle course in training, threading repeatedly the same gaps in the landmarks. Manipulations altering the relations between the landmarks and the surrounding panorama, however, affected the routes in two major ways. Both interchanging the positions of landmarks (transpositions) and displacing the entire landmark set along with the starting position of the ants (translations) (1) reduced the stereotypicality of the route, and (2) increased turns and meanders during travel. The ants might have used the entire panorama in view-based travel, or the distal panorama might prime the identification and use of landmarks en route. Despite the large data set, both options (not mutually exclusive) remain viable.     

S100A2 in cancerogenesis: a friend or a foe?

Owing to the exceptional intracellular distribution and the heterogeneous expression pattern during transformation and metastasis in various tumors, the EF-hand calcium-binding protein S100A2 attracts increasing attention. Unlike the majority of S100 proteins, S100A2 expression is downregulated in many cancers and the loss in nuclear expression has been associated with poor prognosis. On the other hand, S100A2 is upregulated in some cancers. This mini review highlights the general characteristics of S100A2 and discusses recent findings on its putative functional implication as a suppressor or promoter in cancerogenesis.     

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.