γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.     

Synthesis of 2′-Deoxyformycin B and 2′-Deoxyoxoformycin B

Abstract

3-β-D-Ribofuranosylpyazolo[4,3-d]pyrimidines (formycins)¹ modified in the heteroaromatic moiety are of biological interest as analogues of adenosine and guanosine, and have been the objects of intensive synthetic chemical effort by several groups.^2-9 2′-Deoxynucleosides^2c,2d,7b,13 and other analogties of the formycins modified in the sugar moiety^10-12 are also of potential interest, but have been less extensively studied. Examples of the 2′-deoxyribonucleoside type known to date include the 2′-deoxy-6-thioguanosine analogue 1, the 2′-deoxyadenosine (dAdo) analogue 2 (2′-deoxyformycin A),^10,13 and the 2-chloro-2′-deoxyadenosine analogue 3.^7b Compound 2 was found to be 10-15 times more potent than 2′-deoxyadenosine as an inhibitor of the growth of S49 cells, a murine lymphoma line of T-cell origin.¹³ Activity depended on 5′- phosphorylation, since mutants lacking the enzymes adenosine kinase (AK) and deoxycytidine kinase (dCK) were insensitive to the drug. Furthermore, activity was comparable in the presence and absence of an AK inhibitor, suggesting that 2, unlike dAdo, may be a poor substrate for adenosine deaminase. That 5′-phosphorylation of 2 was mediated by AK rather than dCK was indicated by the fact that miitants lacking only dCK retained sensitivity. This contrasted with the behavior of dAdo, which is known to be n substrate for both AK and dCK.¹⁴     

Synthesis and bioevaluation of ω-N-amino analogs of B13

Novel ω-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015].Representative analog LCL464, (1R,2R)-2-N-(12′-N,N-dimethylaminododecanoyl amino)-1-(4″-nitrophenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of sphingosine and specific increase of C₁₄- and C₁₆-ceramide. LCL464 did not induce lysosomal destabilization nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activation. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of ACDase.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Cytogenetic analysis of the 2B3-4 — 2B11 region of the X chromosome of Drosophila melanogaster

Puffing patterns have been studied both in homozygotes t10/t10, a gene located in the area of the early ecdysone puff 2B5, and in a yellow (y) control stock, at the end of the third instar and during prepupal development. In mutants t10 at the end of the third instar puffing develops normally in general, however, 21 puffs (5 early and 16 late ones) underdevelop or do not develop at all, some larval intermoult puffs regressing slower. The next cycle of puffs (mid prepupal) in mutants t10 proceeds normally, but in the late prepupal cycle 21 puffs underdevelop again or are not formed at all. A model for the induction of early ecdysone puffs is proposed, assigning a key role to the 2B5 puff product in stimulating other early puffs. It is suggested that defects in the activity of early puffs in the mutant t10 may cause underdevelopment of late puffs.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Phenotypic Characterization of Autoreactive B Cells—Checkpoints of B Cell Tolerance in Patients with Systemic Lupus Erythematosus

DNA-reactive B cells play a central role in systemic lupus erythematosus (SLE); DNA antibodies precede clinical disease and in established disease correlate with renal inflammation and contribute to dendritic cell activation and high levels of type 1 interferon. A number of central and peripheral B cell tolerance mechanisms designed to control the survival, differentiation and activation of autoreactive B cells are thought to be disturbed in patients with SLE. The characterization of DNA-reactive B cells has, however, been limited by their low frequency in peripheral blood. Using a tetrameric configuration of a peptide mimetope of DNA bound by pathogenic anti-DNA antibodies, we can identify B cells producing potentially pathogenic DNA-reactive antibodies. We, therefore, characterized the maturation and differentiation states of peptide, (ds) double stranded DNA cross-reactive B cells in the peripheral blood of lupus patients and correlated these with clinical disease activity. Flow cytometric analysis demonstrated a significantly higher frequency of tetramer-binding B cells in SLE patients compared to healthy controls. We demonstrated the existence of a novel tolerance checkpoint at the transition of antigen-naïve to antigen-experienced. We further demonstrate that patients with moderately active disease have more autoreactive B cells in both the antigen-naïve and antigen-experienced compartments consistent with greater impairment in B cell tolerance in both early and late checkpoints in these patients than in patients with quiescent disease. This methodology enables us to gain insight into the development and fate of DNA-reactive B cells in individual patients with SLE and paves the way ultimately to permit better and more customized therapies.     

Inhibition of I��B Kinase by Vaccinia Virus Virulence Factor B14

The IκB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-κB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-κB activation in response to TNFα, IL-1β, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vΔB14) there was a higher level of phosphorylated IκBα but a similar level of IκBα compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKβ but not IKKα, suggesting the interaction occurs via IKKβ. B14 inhibited NF-κB activation induced by overexpression of IKKα, IKKβ, and a constitutively active mutant of IKKα, S176/180E, but did not inhibit a comparable mutant of IKKβ, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKβ is targeted by B14, and this was confirmed using Ab specific for phospho-IKKβ.