The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.     

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Housekeeping genes in cancer: normalization of array data

Biological maintenance of cells under variable conditions should affect gene expression of only certain genes while leaving the rest unchanged. The latter, termed "housekeeping genes," by definition must reflect no change in their expression levels during cell development, treatment, or disease state anomalies. However, deviations from this rule have been observed. Using DNA microarray technology, we report here variations in expression levels of certain housekeeping genes in prostate cancer and a colorectal cancer gene therapy model system. To highlight, differential expression was observed for ribosomal protein genes in the prostate cancer cells and beta-actin in treated colorectal cells. High-throughput differential gene expression analysis via microarray technology and quantitative PCR has become a common platform for classifying variations in similar types of cancers, response to chemotherapy, identifying disease markers, etc. Therefore, normalization of the system based on housekeeping genes, such as those reported here in cancer, must be approached with caution.     

On the use of infinite elements for the determination of optimal closure patterns based on stress analysis

Solid infinite elements are used in conjunction with finite elements to compute the stress and displacement distribution resulting from the suturing of wounds of symmetric and nonsymmetric shapes in orthotropic, abdominal human skin. The optimal pattern of suturing of wounds are investigated from a stress perspective. Highly accurate, quantitative and qualitative improvements over the use of finite elements to approximate distant boundaries are obtained. Numerical results quantitatively agree with analytic results computed using complex analysis techniques. The technique used and the results obtained will aid surgeons in closing nonsymmetrical wounds on regions of the body that exhibit orthotropy.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.