Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor.

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ±â€¯0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ±â€¯0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-ß-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-ß-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.

The effect of physicochemical factors on the self-association of HMGB1: A surface plasmon resonance study.

HMGB1 triggers proinflammatory reactions by interacting extracellularly with various receptors. HMGB1 also acts in the nucleus by interacting with DNA and controlling DNA transcription, a process which involves its self-association. The self-association of HMGB1 was characterized using surface plasmon resonance (SPR). A dimer/tetramer binding model was developed that provided a good fit to the SPR sensorgrams and enabled the kinetics of self-association of different HMGB1 oligomers to be evaluated under a variety of physicochemical conditions. The formation of HMGB1 tetramers, and not dimers, was strongly influenced by ionic strength. HMGB1 self-association increased as the pH was decreased from 7.4 to 4.8 but was abolished at pH4.0, suggesting the involvement of acidic amino acids of HMGB1 in its self-association. HMGB1 dimers were found to predominate in the absence of zinc, but addition of zinc promoted the formation of HMGB1 tetramers. More reducing conditions favored dimerization but diminished tetramer formation. In contrast, oxidizing conditions favored tetramer formation. Physicochemical factors modulate the extent of self-association of HMGB1. We speculate that HMGB1 dimers may preferentially bind DNA, whereas HMGB1 tetramers may promote inflammatory responses by binding to RAGE and TLRs. The self-association of HMGB1, regulated by variations of physicochemical factors, may influence its roles in DNA rearrangement and regulation of pathophysiological diseases.

Optimization of surface plasmon resonance experiments: Case of high mobility group box 1 (HMGB1) interactions.

Surface plasmon resonance (SPR) is a powerful technique for evaluating protein-protein interactions in real time. However, inappropriately optimized experiments can often lead to problems in the interpretation of data, leading to unreliable kinetic constants and binding models. Optimization of SPR experiments involving "sticky" proteins, or proteins that tend to aggregate, represents a typical scenario where it is important to minimize errors in the data and the kinetic analysis of those data. This is the case of High Mobility Group Box 1 and the receptor of advanced glycation end products. A number of improvements in protein purification, buffer composition, immobilization conditions, and the choice of flow rate are shown to result in substantial improvements in the accurate characterization of the interactions of these proteins and the derivation of the corresponding kinetic constants.

Predictive mutational bioinformatic analysis of variation in the skin and wool associated corneodesmosin (CDSN) gene in sheep.

Corneodesmosin (CDSN) is an important component of the desmosome in the epidermal cornified stratum and inner root sheath of hair follicles. DNA from a sheep BAC clone previously identified by us to contain CDSN was PCR amplified using cattle-derived primers and the product sequenced. A region of 4579 bp containing CDSN was shown to contain two exons separated by one intron and spanning 3683 bp. The DNA encodes a predicted protein of 546 amino acids. Phylogenetic analysis shows that sheep CDSN falls within a clade containing cattle and other ruminant-like species. Comparison of sequences generated from 12 unrelated merino sheep and the International Sheep Genome Consortium (ISGC) data identified 58 single nucleotide polymorphisms (SNPs) within the 4579 bp region of which 16 are contained within coding sequences (1 in 80 bp). The SNPs identified in this study will add to the Major Histocompatibility Complex (MHC) SNP panel, which will allow extensive haplotyping of the sheep MHC in future studies.

A participatory learning approach to biochemistry using student authored and evaluated multiple-choice questions.

A participatory learning approach, combined with both a traditional and a competitive assessment, was used to motivate students and promote a deep approach to learning biochemistry. Students were challenged to research, author, and explain their own multiple-choice questions (MCQs). They were also required to answer, evaluate, and discuss MCQs written by their peers. The technology used to support this activity was PeerWise--a freely available, innovative web-based system that supports students in the creation of an annotated question repository. In this case study, we describe students' contributions to, and perceptions of, the PeerWise system for a cohort of 107 second-year biomedical science students from three degree streams studying a core biochemistry subject. Our study suggests that the students are eager participants and produce a large repository of relevant, good quality MCQs. In addition, they rate the PeerWise system highly and use higher order thinking skills while taking an active role in their learning. We also discuss potential issues and future work using PeerWise for biomedical students.

Genome sequence and identification of candidate vaccine antigens from the animal pathogen Dichelobacter nodosus.

Dichelobacter nodosus causes ovine footrot, a disease that leads to severe economic losses in the wool and meat industries. We sequenced its 1.4-Mb genome, the smallest known genome of an anaerobe. It differs markedly from small genomes of intracellular bacteria, retaining greater biosynthetic capabilities and lacking any evidence of extensive ongoing genome reduction. Comparative genomic microarray studies and bioinformatic analysis suggested that, despite its small size, almost 20% of the genome is derived from lateral gene transfer. Most of these regions seem to be associated with virulence. Metabolic reconstruction indicated unsuspected capabilities, including carbohydrate utilization, electron transfer and several aerobic pathways. Global transcriptional profiling and bioinformatic analysis enabled the prediction of virulence factors and cell surface proteins. Screening of these proteins against ovine antisera identified eight immunogenic proteins that are candidate antigens for a cross-protective vaccine.

jAMVLE, A new integrated molecular visualization learning environment*.

A new computer-based molecular visualization tool has been developed for teaching, and learning, molecular structure. This java-based jmol Amalgamated Molecular Visualization Learning Environment (jAMVLE) is platform-independent, integrated, and interactive. It has an overall graphical user interface that is intuitive and easy to use. The application can be downloaded free from the internet at wabri.org.au/jamvle. A cohort of 28 third year undergraduate molecular biotechnology degree students evaluated the new application through an essay-style project. These were analyzed to identify themes expressed by students in the content of their evaluations. Most students were positive about the new jAMVLE learning environment, and five major benefits emerged from the analysis. In particular, the students perceived that jAMVLE has an appealing interface, is interactive, provides a useful integrated environment, is user friendly, and is an excellent learning tool. Overall, students found that the jAMVLE application stimulated their interest, was a more active learning environment, provided better guidance, and made learning fun.