RTKN2 induces NF-KappaB dependent resistance to intrinsic apoptosis in HEK cells and REgulates BCL-2 genes in human CD4+ lymphocytes

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4+ T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNFα. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4+ T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4+ T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.

Discovering the effects of omega-3 and omega-6 fatty acids on allergy using a hek-blue cell line

In recent times, allergy has become a financial, physical andpsychological burden to the society as a whole. Allergic reactions can result in life-threatening situations causing morbidity and high economic cost. Therefore, more effective reagents are needed for allergy treatment. Literature suggests that a causal relationship exists between the intake of Omega-3/6 fatty acids such as DHA, EPA, DPA and AA and atopic individuals suffering from allergies. In an allergic cascade, cytokines IL-4 and IL-13 bind to IL-4 receptor (IL-4R), which activates the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE production by B cells. The overall aim of this study is to characterise Omega-3/6 fatty acids and their effects on IgE production.

Discovering the effects of omega-6 fatty acids on allergy using a HEK-Blue cell line

In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. Allergic reactions can result in life-threatening situations causing morbidity and high economic cost. Therefore, more effective reagents are needed for allergy treatment. Omega-6 fatty acids have gained attention in allergic studies mainly due to their inflammatory properties. Literature suggests that a causal relationship exists between the intake of omega-6 fatty acids such as DPA and AA and atopic individuals suffering from allergies. In an allergic cascade, cytokines IL-4 and IL-13 bind to IL-4 receptor (IL-4R), which activates the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE production by B cells. Consequently, IgE production leads to clinical symptoms of allergy. The overall aim of this study is to characterise DPA and AA and their effects on IgE production.

Exploring the effects of omega-3 and omega-6 fatty Acids on allergy using a HEK-blue cell line.

BACKGROUND: Allergic reactions can result in life-threatening situations resulting in high economic costs and morbidity. Therefore, more effective reagents are needed for allergy treatment. A causal relationship has been suggested to exist between the intake of omega-3/6 fatty acids, such as docosahexanoic acid (DHA), eicosapentanoic acid (EPA), docosapentanoic acid (DPA) and arachidonic acid (AA), and atopic individuals suffering from allergies. In allergic cascades, the hallmark cytokine IL-4 bind to IL-4 receptor (IL-4R) and IL-13 binds to IL-13 receptor (IL-13R), this activates the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE antibody production by B cells. The overall aim of this study was to characterize omega-3/6 fatty acids and their effects on STAT6 signaling pathway that results in IgE production in allergic individuals. METHODS: The fatty acids were tested in vitro with a HEK-Blue IL-4/IL-13 reporter cell line model, transfected with a reporter gene that produces an enzyme, secreted embryonic alkaline phosphatase (SEAP). SEAP acts as a substitute to IgE when cells are stimulated with bioactive cytokines IL-4 and/or IL-13. RESULTS: We have successfully used DHA, EPA and DPA in our studies that demonstrated a decrease in SEAP secretion, as opposed to an increase in SEAP secretion with AA treatment. A statistical Student's t-test revealed the significance of the results, confirming our initial hypothesis. CONCLUSION: We have successfully identified and characterised DHA, EPA, DPA and AA in our allergy model. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors of IL-4R/IL-13R signalling, which regulates the STAT6 induced pathway in allergic cascades. Such findings are significant in the future design of dietary therapeutics for the treatment of allergies.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

Isolation breeds naivety : island living robs Australian varanid lizards of toad-toxin immunity via four-base-pair mutation

Since their introduction to the toad-free Australian continent cane toads (Bufo marinus) have caused a dramatic increase in naïve varanid mortality when these large lizards attempt to feed on this toxic amphibian. In contrast Asian–African varanids, which have coevolved with toads, are resistant to toad toxin. Toad toxins, such as Bufalin target the H1-H2 domain of the α1 subunit of the sodium-potassium-ATPase enzyme. Sequencing of this domain revealed identical nucleotide sequences in four Asian as well as in three African varanids, and identical sequences in all 11 Australian varanids. However, compared to the Asian–African varanids, the Australian varanids showed four-base-pair substitutions, resulting in the alteration in three of the 12 amino acids representing the H1-H2 domain. The phenotypic effect of the substitutions was investigated in human embryonic kidney (HEK) 293 cells stably transfected with the Australian and the Asian–African H1-H2 domains. The transfections resulted in an approximate 3000-fold reduction in resistance to Bufalin in the Australian HEK293 cells compared to the Asian–African HEK293 cells, demonstrating the critical role of this minor mutation in providing Bufalin resistance. Our study hence presents a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.

Identification and characterization of a novel IL-4 receptor α chain (IL-4Rα) antagonist to inhibit IL-4 signalling

BACKGROUND/AIMS: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R), consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. METHODS: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. RESULTS: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP). QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. CONCLUSION: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

Book review: An Appreciation of Difference: WEH Stanner and Aboriginal Australia, edited by Melinda Hinkson and Jeremy Beckett, xvii + 293 pp, Aboriginal Studies Press, Canberra, 2008, ISBN 9780855756604 (pbk), $39.95

An Appreciation of Difference: WEHStanner and Aboriginal Australia is a tribute nearly 30 years after his death to one of the lost esteemed anthropologists who worked in Indigenous Australian contexts. It is also a reflection on what Stanner achieved in his lifetime and what his work contributes to current Indigenous issues and Indigenous studies in Australia today.