Cholesterol modification of proteins

The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning. Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol. Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins. We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues. Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal.     

Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.     

Engineering secretable forms of chaperones for immune modulation and vaccine development

Heat shock proteins are present in almost all intracellular compartments and serve by folding newly synthesized proteins, disassembling unstable proteins, and assisting in the transportation of proteins within the cell. Under certain circumstances they are also present on the cell surface, and can be shed or secreted into the extracellular environment. Although they possess many functional roles, their ability to stimulate innate and antigen-specific immunity have made them attractive candidates for vaccine development. Here, we review some of the approaches that have been used to genetically engineer molecular chaperones for their secretion from tumor cells or targeting them to the plasma membrane of such cells in order to promote anti-tumor responses. Treatment of tumor cells engineered to secrete or display chaperones may be of benefit, particularly in the area of cell-based vaccine development.     

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV)

Plasmodesmatal gating in epidermal cells of Nicotianatabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic ‘halo’ shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.     

Tissue-specific and temporal regulation of a β-conglycinin gene: roles of the RY repeat and other cis-acting elements

Upstream regulatory sequences (URS) of the gene that encodes the subunit of -conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623–631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.