Chiral linear hydroxamates as biomimetic analogues of ferrioxamine and coprogen and their use in probing siderophore-receptor specificity in bacteria and fungi

Summary Linear hydroxamate derivatives, possessing chiral -amino acid moieties, were synthesized and their iron transport activities were studied in bacteria and fungi. No growth-promoting activity could be detected in the Gram-positive hydroxamate-auxotrophAureobacterium flavescens JG9. However, Gram-negative enterobacteria, such asEscherichia coli, Pantoea agglomerans andHafnia alvei were able to utilize iron from these analogues. Uptake of⁵⁵Fe-labeled analogues was inhibited by sodium azide, suggesting an active transport process. The receptors involved during uptake in enterobacteria were identified by using appropriate indicator organisms which are defective in the transport of either ferrioxamines (P. agglomerans FM13), coprogens (H. alvei), or both of these siderophore classes (E. coli fhuE). Our data suggest that the chiral hydroxamates are recognized by the ferrioxamine receptor (FoxA) and the coprogen receptor (FhuE) at a ratio which depends on the optical/ isomer fraction and the nature of side chains. Transport was also observed in the fungusNeurospora crassa, known to take up coprogen rather than ferrioxamines, suggesting that in this fungus the synthetic analogues behave like coprogen.     

DNA vaccination with heat shock protein 60 inhibits cyclophosphamide-accelerated diabetes

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-gamma secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of beta cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.     

A3 adenosine receptor activation in melanoma cells: association between receptor fate and tumor growth inhibition

Activation of the Gi protein-coupled A3 adenosine receptor (A3AR) has been implicated in the inhibition of melanoma cell growth by deregulating protein kinase A and key components of the Wnt signaling pathway. Receptor activation results in internalization/recycling events that play an important role in turning on/off receptor-mediated signal transduction pathways. Thus, we hereby examined the association between receptor fate, receptor functionality, and tumor growth inhibition upon activation with the agonist 1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purine-9-yl]-N-methyl-beta-D-ribofuranuronamide (IB-MECA). Results showed that melanoma cells highly expressed A3AR on the cell surface, which was rapidly internalized to the cytosol and "sorted" to the endosomes for recycling and to the lysosomes for degradation. Receptor distribution in the lysosomes was consistent with the down-regulation of receptor protein expression and was followed by mRNA and protein resynthesis. At each stage, receptor functionality was evidenced by the modulation in cAMP level and the downstream effectors protein kinase A, glycogen synthase kinase-3beta, c-Myc, and cyclin D1. The A3AR antagonist MRS 1523 counteracted the internalization process as well as the modulation in the expression of the signaling proteins, demonstrating that the responses are A3AR-mediated. Supporting this notion are the in vivo studies showing tumor growth inhibition upon IB-MECA treatment and reverse of this response when IB-MECA was given in combination with MRS 1523. In addition, in melanoma tumor lesions derived from IB-MECA-treated mice, the expression level A3AR and the downstream key signaling proteins were modulated in the same pattern as was seen in vitro. Altogether, our observations tie the fate of A3AR to modulation of downstream molecular mechanisms leading to tumor growth inhibition both in vitro and in vivo.     

Susceptibility Loci Associated with Specific and Shared Subtypes of Lymphoid Malignancies

The genetics of lymphoma susceptibility reflect the marked heterogeneity of diseases that comprise this broad phenotype. However, multiple subtypes of lymphoma are observed in some families, suggesting shared pathways of genetic predisposition to these pathologically distinct entities. Using a two-stage GWAS, we tested 530,583 SNPs in 944 cases of lymphoma, including 282 familial cases, and 4,044 public shared controls, followed by genotyping of 50 SNPs in 1,245 cases and 2,596 controls. A novel region on 11q12.1 showed association with combined lymphoma (LYM) subtypes. SNPs in this region included rs12289961 near LPXN, (P_LYM = 3.89×10⁻⁸, OR = 1.29) and rs948562 (P_LYM = 5.85×10⁻⁷, OR = 1.29). A SNP in a novel non-HLA region on 6p23 (rs707824, P_NHL = 5.72×10⁻⁷) was suggestive of an association conferring susceptibility to lymphoma. Four SNPs, all in a previously reported HLA region, 6p21.32, showed genome-wide significant associations with follicular lymphoma. The most significant association with follicular lymphoma was for rs4530903 (P_FL = 2.69×10⁻¹², OR = 1.93). Three novel SNPs near the HLA locus, rs9268853, rs2647046, and rs2621416, demonstrated additional variation contributing toward genetic susceptibility to FL associated with this region. Genes implicated by GWAS were also found to be cis-eQTLs in lymphoblastoid cell lines; candidate genes in these regions have been implicated in hematopoiesis and immune function. These results, showing novel susceptibility regions and allelic heterogeneity, point to the existence of pathways of susceptibility to both shared as well as specific subtypes of lymphoid malignancy.     

Displaced Enemies,Displaced Memories: Diaspora Memorial Politics of Partition and the Holocaust

This paper argues that the refusal of the Muslim Council of Britain to attend Holocaust Memorial Day highlights a key dimension of memory as political myth: namely, the sense that time is cyclical. Prior external and internal enemies (in their current manifestations) are apocalyptically destined to threaten the integrity of the nation once more. Hence, ideologies based on political myths draw on both the future hopes and the future fears of people. The paper highlights the similarities between Jewish and Pakistani fears, rooted in the Holocaust and Partition, of a repeated ‘cycle of death and suffering’. These loom large especially for those suffering racism. The more bound people are by their narrow group's particular symbols and history, the more apocalyptic their vision of this future is likely to be.     

Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.     

Intraamniotic Ethyl Docosahexaenoate Administration Protects Fetal Rat Brain from Ischemic Stress

Abstract: Studies were conducted on the prenatal rat given a single intraamniotic injection of ethyl docosahexaenoate (Et-DHA; 9.6–12 mmol per fetus) or subjected to an n-3 fatty acid-deficient diet to assess the role of docosahexaenoate on oxidative stress during episodes of ischemia. A time-dependent decrease in the ability of brain slices from animals treated with Et-DHA to produce thiobarbituric acid-reactive substance (TBARS), most pronounced after 1 day (from 58.1 ± 4.22 to 15.9 ± 1.6 nmol/mg of DNA), was noticed on stimulation with Fe²⁺. Brain slices from fetuses treated for 1 day with Et-DHA and those from untreated fetuses produced TBARS levels of 46.7 ± 6.5 and 114.8 ± 10.8 nmol/mg of DNA, respectively, after a 20-min occlusion of the fetal-maternal circulation at embryonic day 20, suggesting a protective effect of Et-DHA. The protective effect of a single dose of Et-DHA in utero remained high up to 3 days after injection ( p < 0.001) and was long-lasting, yet not significant, up to 3 days following birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et-DHA-treated animals were lower than in the controls. Et-DHA-injected fetuses exhibited significantly higher levels of esterified DHA than the non-injected controls. n-3-deficient diet given to dams for 2 weeks before birth did not affect the levels of TBARS production in control fetal brain slices but abolished the increase caused by ischemia. Et-DHA administration for 24 h to n-3-deficient fetuses reduced the amount of TBARS produced by the fetal brain slices from 49.1 ± 8.5 to 31.7 ± 4.1 nmol/mg of DNA. A protective effect from oxidative damage after postischemic oxidative stress in fetal brain following DHA supplements is suggested, whereas the effect of n-3 fatty acid deficiency in this regard is more ambiguous.