Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala⁷⁶:Val⁷⁷ within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.     

Accurately Measuring Recombination between Closely Related HIV-1 Genomes

Retroviral recombination is thought to play an important role in the generation of immune escape and multiple drug resistance by shuffling pre-existing mutations in the viral population. Current estimates of HIV-1 recombination rates are derived from measurements within reporter gene sequences or genetically divergent HIV sequences. These measurements do not mimic the recombination occurring in vivo, between closely related genomes. Additionally, the methods used to measure recombination make a variety of assumptions about the underlying process, and often fail to account adequately for issues such as co-infection of cells or the possibility of multiple template switches between recombination sites. We have developed a HIV-1 marker system by making a small number of codon modifications in gag which allow recombination to be measured over various lengths between closely related viral genomes. We have developed statistical tools to measure recombination rates that can compensate for the possibility of multiple template switches. Our results show that when multiple template switches are ignored the error is substantial, particularly when recombination rates are high, or the genomic distance is large. We demonstrate that this system is applicable to other studies to accurately measure the recombination rate and show that recombination does not occur randomly within the HIV genome.     

Characterisation of the PTEN inhibitor VO-OHpic

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K _m and V _max are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K _ic and K _iu were determined to be 27 ± 6 and 45 ± 11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP₃) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening.     

A proteomic approach to the identification and characterisation of protein composition in wheat germ

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0–7.0 and 6.0–9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.     

The effects of dexamethasone and dehydroepiandrosterone (DHEA) on cytokines and receptor expression in a human osteoblastic cell line: potential steroid-sparing role for DHEA

Osteoporosis and associated fractures are the most common and debilitating complication of glucocorticoid use. The use of alternative anti-inflammatory agents without the deleterious skeletal effects of glucocorticoids is needed. Dehydroepiandrosterone (DHEA) may have immunomodulatory as well as positive effects on bone. For our further understanding of the mechanisms of action of DHEA, as a steroid-sparing agent, we investigated and compared the effects of dexamethasone (DEX) and DHEA on the regulation of the downstream effector pathway of osteoclastogenesis; RANKL/OPG and a range of inflammatory/pro-resorbing cytokines and receptors using a human clonal osteoblastic cell line. The cells were treated with DEX, DHEA, and androstenedione (ANDI). The mRNA expression of RANKL and OPG was determined by real-time PCR after overnight incubation. The regulation of a broad spectrum of cytokines by DEX and DHEA was also investigated using a human cytokine/growth factor and receptor gene array consisting of 268 cytokine-related cDNAs. To confirm some of the gene expression changes, protein production was measured by ELISA. RANKL expression and RANKL/OPG ratio were increased by DEX. This effect was reversed by co-treatment with both DHEA or ANDI. Several pro-inflammatory/resorptive cytokines including IL-6, IL-4, IFN-gamma, macrophage inhibitory factor (MIF) were down-regulated not only by DEX but also by DHEA. In contrast to DEX, DHEA did not lead to suppression of growth factors including vascular endothelial growth factor (VEGF), fibroblast growth factor-5 (FGF-5), insulin-like growth factor-binding protein3 (IGF-BP3). Several new target genes previously documented to influence bone formation were up-regulated by DHEA such as Notch 2, insulin receptor, thrombin receptor (PAR1). The data suggest that DHEA has immunomodulatory properties without the catabolic effects on bone remodeling, observed with glucocorticoid use. DHEA may thus prove useful as a steroid-sparing agent in the management of inflammatory disorders such as SLE or rheumatoid arthritis. Further in vivo studies are indicated.     

PTEN in the haematopoietic system and its therapeutic indications

A unique feature of the haematopoietic system is its self-renewal ability while maintaining a stable number of pluripotent haematopoietic stem cells (HSCs). Recently, two publications by Yilmaz and colleagues and Zhang and colleagues demonstrated that the loss of the tumour suppressor phosphatase and tensin homolog (PTEN) in mice disturbed the maintenance of quiescent HSCs and promoted leukemogenesis. Mammalian target of rapamycin (mTOR) inhibition with rapamycin distinctly rescued HSC development and depleted leukemic stem cells. Thus, the regulation of HSCs and leukemic cells seems to be governed by cell-context-dependent, PTEN-mediated regulation of mTOR.     

Effects of Ginkgolide on the development of NOS and AChE positive neurons in the embryonic basal forebrain

Extract of Ginkgo biloba (EGb) has been therapeutically used for several decades to increase peripheral and cerebral blood flow so as to prevent cardiovascular and neurovascular diseases. However, the role of EGb in neuroprotective effects has received much attention recently. In this study, we investigated the effect of EGb on the development of NOS and AChE positive neurons in the rat embryonic basal forebrain. The results showed that treated with EGb, the OD of MTT staining analysis, and the numbers, the cell sizes and circumferences of NOS and AChE positive neurons were greatly promoted. These data suggest that EGb had similar effects of the neurotrophins such as NGF and BDNF in promoting the development of NOS and AChE positive neurons in the rat embryonic basal forebrain.