Stat3 Signaling Promotes Survival And Maintenance Of Medullary Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) are essential for establishing central tolerance by expressing a diverse array of self-peptides that delete autoreactive thymocytes and/or divert thymocytes into the regulatory T cell lineage. Activation of the NFκB signaling pathway in mTEC precursors is indispensable for mTEC maturation and proliferation resulting in proper medullary region formation. Here we show that the Stat3-mediated signaling pathway also plays a key role in mTEC development and homeostasis. Expression of a constitutively active Stat3 transgene targeted to the mTEC compartment increases mTEC cellularity and bypasses the requirement for signals from positively selected thymocytes to drive medullary region formation. Conversely, conditional deletion of Stat3 disrupts medullary region architecture and reduces the number of mTECs. Stat3 signaling does not affect mTEC proliferation, but rather promotes survival of immature MHCII^loCD80^lo mTEC precursors. In contrast to striking alterations in the mTEC compartment, neither enforced expression nor deletion of Stat3 affects cTEC cellularity or organization. These results demonstrate that in addition to the NFkB pathway, Stat3-mediated signals play an essential role in regulating mTEC cellularity and medullary region homeostasis.     

Thymus medulla formation and central tolerance are restored in IKKalpha-/- mice that express an IKKalpha transgene in keratin 5+ thymic epithelial cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-kappaB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IkappaB kinase (IKK)alpha, a required intermediate in the noncanonical NF-kappaB signaling pathway. Introduction of an IKKalpha transgene driven by a K5 promoter restores the K5(+)K14(+) mTEC subset in IKKalpha(-/-) mice. Unexpectedly, the K5-IKKalpha transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKalpha transgene ameliorates autoimmune symptoms in IKKalpha(-/-) mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-kappaB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.     

Molecular docking and simulation of Curcumin with Geranylgeranyl Transferase1 (GGTase1) and Farnesyl Transferase (FTase)

Protein prenylation is a posttranslational modification that is indispensable for translocation of membrane GTPases like Ras, Rho, Ras etc. Proteins of Ras family undergo farnesylation by FTase while Rho family goes through geranylgeranylation by GGTase1. There is only an infinitesimal difference in signal recognition between FTase and GGTase1. FTase inhibitors mostly end up selecting the cells with mutated Ras proteins that have acquired affinity towards GGTase1 in cancer microcosms. Therefore, it is of interest to identify GGTase1 and FTase dual inhibitors using the docking tool AutoDock Vina. Docking data show that curcumin (from turmeric) has higher binding affinity to GGTase1 than that of established peptidomimetic GGTase1 inhibitors (GGTI) such as GGTI-297, GGTI-298, CHEMBL525185. Curcumin also interacts with FTase with binding energy comparable to co-crystalized compound 2-[3-(3-ethyl-1-methyl-2-oxo-azepan-3-yl)-phenoxy]-4-[1-amino-1-(1-methyl-1h-imidizol-5-yl)-ethyl]-benzonitrile (BNE). The docked complex was further simulated for 10 ns using molecular dynamics simulation for stability. Thus, the molecular basis for curcumin binding to GGTase1 and FTase is reported.     

24-hour rhythms in oxidative stress during hepatocarcinogenesis in rats: effect of melatonin or alpha-ketoglutarate

To compare the effects of alpha-ketoglutarate (alpha-KG) and melatonin on 24-h rhythmicity of oxidative stress in N-nitrosodiethylamine (NDEA)-injected Wistar male rats, melatonin (5 mg/kg i.p.) or alpha-KG (2 g/kg through an intragastric tube) was given daily for 20 weeks. In blood collected at 6 time points during a 24-h period, serum activity of aspartate transaminase (AST) and alanine transaminase (ALT) and the levels of alpha-fetoprotein (alpha-FP) were measured as markers of liver function. To assess lipid peroxidation and the antioxidant status, plasma levels of thiobarbituric acid reactive substances (TBARS) and of reduced glutathione (GSH) were measured, together with the activity of erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST). NDEA augmented mesor and amplitude of rhythms in AST and ALT activity and plasma alpha-FP levels and mesor values of plasma TBARS, while decreasing mesor values of plasma GSH and erythrocyte SOD, CAT, GPx and GST. Acrophases were delayed by NDEA in all cases except for alpha-FP rhythm, which became phase-advanced. Co-administration of melatonin or alpha-KG partially counteracted the effects of NDEA. Melatonin decreased mesor of plasma TBARS and augmented mesor of SOD activity. The results indicate that melatonin and alpha-KG are effective in protecting from NDEA-induced perturbation of 24-h rhythms in oxidative stress. Melatonin augmented antioxidant defense in rats.     

Section: bioprocessing and biological engineering gene fragment polymerization for increased yield of recombinant HIV fusion inhibitor enfuvirtide

Fuzeon (Enfuvirtide, T20) is the first fusion inhibitor approved by the FDA of the USA for the treatment of HIV/AIDS in combination with other anti-retroviral drugs. Enfuvirtide is a synthetic peptide that blocks the entry of HIV into healthy host CD₄ cells, which requires very high (90 mg twice daily) therapeutic doses. To increase the yield of Enfuvirtide, a gene polymerization strategy was introduced and recombinant T20 (rT20) was expressed in Escherichia coli as a five copy repeat polypeptide with a histidine-tag. The five copy rT20 was purified by Ni-affinity chromatography and cleaved to single rT20 units by cyanogen bromide. Finally, single rT20 units were purified by reversed phase chromatography giving a yield (400 mg/l) with a purity >95 %, which exhibited specific biological activity similar to Fuzeon.     

Ornithine alpha-ketoglutarate modulates the levels of antioxidants and lipid peroxidation products in ammonium acetate treated rats

The effects of ornithine alpha-ketoglutarate (OKG) on ammonium acetate induced hepatotoxicity were studied in experimental rats. The levels of urea, non-protein nitrogen and thiobarbituric acid reactive substances were significantly increased in ammonium acetate treated rats; but these levels were significantly decreased in ammonium acetate-OKG treated rats. Similar patterns were observed in the levels of free fatty acids, triglycerides and phospholipids. Furthermore, non-enzymatic (reduced glutathione) and enzymatic (glutathione peroxidase, superoxide dismutase and catalase) antioxidants were significantly decreased in ammonium acetate treated rats, when compared with control and were significantly increased in ammonium acetate-OKG treated rats compared to ammonium acetate treatment alone.     

A Homology Based Model and Virtual Screening of Inhibitors for Human Geranylgeranyl Transferase 1 (GGTase1)

Protein prenylation is a post translational modification that is indispensable for Ras–Rho mediated tumorigenesis. In mammals, three enzymes namely protein farnesyltransferase (FTase), geranylgeranyl transferase1 (GGTase1), and geranylgeranyl transferase2 (GGTase2) were found to be involved in this process. Usually proteins of Ras family will be farnesylated by FTase, Rho family will be geranylgeranylated by GGTase1. GGTase2 is exclusive for geranylgeranylating Rab protein family. FTase inhibitors such as FTI- 277 are potent anti-cancer agents in vitro. In vivo, mutated Ras proteins can either improve their affinity for FTase active site or undergo geranylgeranylation which confers resistance and no activity of FTase inhibitors. This led to the development of GGTase1 inhibitors. A well-defined 3-D structure of human GGTase1 protein is lacking which impairs its in silico and rational designing of inhibitors. A 3-D structure of human GGTase1 was constructed based on primary sequence available and homology modeling to which pubchem molecules library was virtually screened through AutoDock Vina. Our studies show that natural compounds Camptothecin (-8.2 Kcal/mol), Curcumin (-7.3 Kcal/mol) have higher binding affinities to GGTase-1 than that of established peptidomimetic GGTase-1 inhibitors such as GGTI-297 (-7.5 Kcal/mol), GGTI-298 (-7.5 Kcal/mol), CHEMBL525185 (-7.2 Kcal/mol).     

Protective influences of alpha-ketoglutarate on lipid peroxidation and antioxidant status in ammonium acetate treated rats

The effects of alpha-ketoglutarate on ammonium acetate induced hyperammonemia were studied biochemically in experimental rats. The levels of circulatory, non-protein nitrogen, serum transaminases and thiobarbituric acid reactive substances were significantly increased in ammonium acetate treated rats. These levels were significantly decreased in alpha-ketoglutarate and ammonium acetate treated rats. Similar patterns of alterations were observed in the levels of free fatty acids, triglycerides, phopholipids and cholesterol inbetween various groups. Further non-enzymatic (vitamins C and E) and enzymatic (superoxide dismutase and catalase) antioxidants were significantly decreased in ammonium acetate treated rats; and were significantly increased in alpha-ketoglutarate and ammonium acetate treated rats. The biochemical alterations during alpha-ketoglutarate treatment could be due to (i) the detoxification of excess ammonia, (ii) by participating in the non-enzymatic oxidative decarboxylation in the hydrogen peroxide decomposition process and (iii) by enhancing the proper metabolism of fats which could suppress oxygen radicals generation and thus prevent the lipid peroxidative damages in rats.