Development of ligase-assisted spacer addition for the measurement of microsatellites

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method.     

How do the consumption and development rates of the conifer specialist Aphidecta obliterata respond to temperature,and is it better adapted to limited prey than a generalist?

This study aimed to address the effect of temperature on the consumption and development rates of Aphidecta obliterata and to compare the responses of Ap. obliterata (specialist) with that of Adalia bipunctata (generalist) to prey limitation. Temperature had a significant and positive effect on the time to egg hatch of Ap. obliterata . The duration of the larval instars was not affected by prey species at 15°C but was significantly shorter, 12.1 days at 20°C compared with 21.1 days at 15°C. The proportion of time spent in each instar, however, was not affected by temperature or prey species, but the duration of the pupal stage was significantly affected by temperature. The average daily consumption of prey aphids increased with instar and was significantly influenced by temperature. There was a significant difference in the length of the pupal stage between coccinellid species but not that of the larval stage. The duration of the larval period increased under conditions of prey shortage. The pupal period of Ap. obliterata was significantly affected by the food regime but not that of Ad. bipunctata . There was a significant interaction between species and food supply on the length of the pupal stage and the larval stage and the final fresh weight achieved by the newly emerged adults. Male adults weighed significantly less than the females in all regimes. Larvae of Ap. obliterata and Ad. bipunctata did not consume any of the alternative prey (Collembola or Psocoptera) provided. There was no significant difference in the consumption of prey between the two coccinellid species. The results suggest that both of these coccinellids are well adapted to low-density-specific prey. There were no obvious differences between the two, which would tend to favour either species in an environment of limited prey.     

Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitor

Human thymidine phosphorylase (HTP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), is overexpressed in certain solid tumors where it is linked to poor prognosis. HTP expression is utilized for certain chemotherapeutic strategies and is also thought to play a role in tumor angiogenesis. We determined the structure of HTP bound to the small molecule inhibitor 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI). The inhibitor appears to mimic the substrate transition state, which may help explain the potency of this inhibitor and the catalytic mechanism of pyrimidine nucleotide phosphorylases (PYNPs). Further, we have confirmed the validity of the HTP structure as a template for structure-based drug design by predicting binding affinities for TPI and other known HTP inhibitors using in silico docking techniques. This work provides the first structural insight into the binding mode of any inhibitor to this important drug target and forms the basis for designing novel inhibitors for use in anticancer therapy.     

Intraspecific communication in goldfish

Part I. Characteristics of locomotor behaviour of goldfish ( Carassius auratus ) were monitored automatically and the response to sensory cues provided by conspecifics was analysed. Goldfish were attracted to free-swimming conspecifics and oriented to them klinokinetically by decreasing their mean angle size of turns and increasing both their turning frequency and mean step length between turns. These locomotor changes were stimulus bound. Although the responses were mediated visually, blinded goldfish, which were not overtly attracted, nevertheless oriented orthokinetically to non-visual cues from the conspecific.
Part II. Simulation of the conspecific by models demonstrated that oval and horizontal rectangular shapes of the same area and their size interactions, elicited significant attraction whereas square and vertical rectangular shapes of the same area did not. Increasing speeds and/or size of the model tended to bring about stronger attraction.     

Life-cycle differentiation in Trypanosoma brucei: molecules and mutants

Differentiation between bloodstream and tsetse midgut procyclic forms during the life cycle of the African trypanosome is an attractive model for the analysis of stage-regulated events. In particular, this transformation occurs synchronously, there are well-defined markers for stage-regulated processes and cell lines with specific defects in differentiation have been identified. This combination of tools, combined with the developing Trypanosoma brucei genome database is allowing its underlying controls to be investigated at the molecular and cytological levels. This paper examines some recent discoveries that illuminate some of the key events during trypanosome life-cycle progression.     

Interaction of the release factors with the Escherichia coli ribosome: structurally and functionally-important domains.

There are two major domains of interaction between the Escherichia coli release factors (RF-1 and RF-2) and each subunit of the ribosome. RF-2 has a binding domain on the shoulder and lower head region of the small subunit at the small lobe distant from the decoding site. This is in close proximity to one of the domains on the large subunit which includes the body dimer of L7/L12 and L11. The other domains of interaction, at the decoding site on the small subunit, and at the peptidyltransferase centre of the large subunit of the ribosome, are some distance from the first two, although the evidence for direct contact with the ribosome is less comprehensive. The release factors may therefore have two distinct structural domains, and in support of this concept RF-1 and RF-2 can both be cleaved into two fragments by papain. Region-specific antibodies, and antibodies against defined peptide within the RF sequences have given an indication that a significant part of an interacting RF molecule is in close proximity to the ribosome surface, confirming an observation by immunoelectron microscopy which suggested that the RF penetrates deeply into the cleft between the two subunits. A region of highly conserved primary sequence between the two release factors from E coli is also conserved in those from B subtilis suggesting it forms an important structural or functional domain. Antibodies against peptides from the N-terminal end of this region strongly inhibit binding of the RF to the ribosome.     

The role of smooth muscle in regulating prostatic induction

We have examined the role that smooth muscle plays during prostatic organogenesis and propose that differentiation of a smooth muscle layer regulates prostatic induction by controlling mesenchymal/epithelial interactions. During development of the rat reproductive tract, an area of condensed mesenchyme involved in prostatic organogenesis is formed. This mesenchyme (the ventral mesenchymal pad, VMP) is found in both males and females, yet only males develop a prostate. We demonstrate that a layer of smooth muscle differentiates between the VMP and the urethral epithelium, and that there is a sexually dimorphic difference in the development of this layer. Serial section reconstruction showed that the layer formed at approximately embryonic day 20.5 in females, but did not form in males. In cultures of female reproductive tracts, testosterone was able to regulate the thickness of this layer resulting in a 2.4-fold reduction in thickness. We observed that prostatic buds were present in some female reproductive tracts, and determined that testosterone was able to stimulate prostatic organogenesis, depending upon the bud position relative to the smooth muscle layer. In vitro recombination experiments demonstrated that direct contact with the VMP led to the induction of very few epithelial buds, and that androgens dramatically increased bud development. Taken together, our data suggest that differentiation of a smooth muscle layer regulates signalling between mesenchyme and epithelium, and comprises part of the mechanism regulating prostatic induction.     

KRAS Mutation Analysis by PCR: A Comparison of Two Methods

Background

KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.

Methods

DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer’s instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.

Results

Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.

Conclusion

There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.     

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification

The cyclopentenone prostaglandin (cyPG) PGA₁ displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA₁ derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA₁ (PGA₁-biotinamide, PGA₁-B), to establish its validity to identify cyPG–protein interactions of potential therapeutic interest. PGA₁ and PGA₁-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA₁-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA₁-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA₁-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA₁ that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.