Volume 390, Issue 4

The average amount of cholesterol in the whole animal equals approximately 2100 mg/kg body weight, and 15% and 23% of this sterol in the mouse and human, respectively, is found in the central nervous system. There is no detectable uptake across the blood-brain barrier of cholesterol carried in lipoproteins in the plasma, even in the newborn. However, high rates of de novo cholesterol synthesis in the glia and neurons provide the sterol necessary for early brain development. Once a stable brain size is achieved in the adult, cholesterol synthesis continues, albeit at a much lower rate, and this synthesis is just balanced by the excretion of an equal amount of sterol, either as 24( S )-hydroxycholesterol or, presumably, as cholesterol itself.

Cathelicidins are effector molecules of the innate host defense system that establish an antimicrobial barrier at epithelial interfaces. The human cathelicidin LL-37, in addition to its antimicrobial activity, also exhibits immunomodulatory effects, such as inhibition of pro-inflammatory responses to bacterial LPS in human monocytic cells. In this report, we demonstrate that LL-37 almost completely prevents the pro-inflammatory cytokine release by human peripheral blood mononuclear cells (PBMCs) following stimulation with Toll-like receptor (TLR)4 and TLR2/1 agonists while leaving TLR2/6, TLR5, TLR7 and TLR8 responses unchanged. Modulation of the TLR response by LL-37 occurred at least partly through the MAP kinase pathway via inhibition of p38 phosphorylation. By using an LL-37 library with overlapping sequences, we identified the mid-region of LL-37, comprising amino acids 13–31, as the active domain for the modulation of TLR responses. The mechanism of immunomodulation of LL-37 and LL-37 fragments is lipopoly-saccharide binding. Correlations between the capacity of LL-37 fragments to modulate TLR responses and their physico-chemical properties revealed that cationicity and hydrophobicity are essential for the modulation of LL-37-mediated TLR responses.

Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immunological approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity ( K d =0.58 μ m at 25°C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase ( K d =32 μ m ) and melanoma antigen recognized by T cells (MART-1) ( K d =2.4 μ m ). Adding a peptide sequence (pep70) with high Hsp70 binding affinity ( K d =0.04 μ m ) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation.

Overexpression of human CYP4Z1, a cytochrome P450 enzyme, has been correlated with poor prognosis in human cancer. However, its catalytic properties are not yet known. We expressed this P450 in Schizosaccharomyces pombe and demonstrate by whole-cell biotransformation assays CYP4Z1-dependent in-chain hydroxylation of lauric and myristic acid, which in both cases leads to the formation of four different monohydroxylated products at positions ω-2, ω-3, ω-4, and ω-5, respectively. The CYP4Z1-expressing fission yeast should be a new valuable tool for testing cancer drugs or for the development of new prodrug strategies.

Dengue virus (DENV) and tick-borne encephalitis virus (TBEV) are flaviviruses, which can cause lethal hemorrhagic fever and encephalitis, respectively. Here, we demonstrate that the TBEV-NS5 and DENV-NS5 proteins use an internal binding mechanism to target human PDZ proteins. TBEV-NS5 has high affinity to regulating synaptic membrane exocytosis-2 (RIMS2) and Scribble, whereas DENV-NS5 binds primarily to the tight junction protein zonula occludens-1 (ZO-1). Targeting of TBEV-NS5 to the plasma membrane is stabilised by ZO-1; however, DENV-NS5 co-localises with ZO-1 in the nucleus. These interactions have potential important roles in the ability of flaviviruses to manipulate cell proliferation, junction permeability and the interferon pathways.

Enhanced levels of 9- O- acetylated sialoglycoproteins (Neu5,9Ac 2 GPs) as disease-associated molecules was reported to act as signaling molecules for promoting survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL). Here, we searched for potential physiological ligands for Neu5,9Ac 2 GPs that could be involved in modulating the survival of lymphoblasts. Accordingly, we examined the presence of binding proteins for Neu5,9Ac 2 GPs on cell lines and primary cells of patients with B- and T-ALL, at presentation of the disease. Peripheral blood mononuclear cells from normal healthy donors and cells from myeloid leukemia patients were used for comparison. Neu5,9Ac 2 GPs-binding proteins (BPs) were specifically detected on the surface of both T- and B-ALL-lymphoblasts and ALL-cell lines along with the consistent presence of Neu5,9Ac 2 GPs. The Neu5,9Ac 2 GPs and BPs also co-localized on the cell surface and interacted specifically in vitro . Apoptosis of lymphoblasts, induced by serum starvation, was reversed in the presence of purified Neu5,9Ac 2 GPs due to possible engagement of BPs, and the anti-apoptotic role of this interaction was established. This is the first report of the presence of potential physiological ligands for disease-associated molecules like Neu5,9Ac 2 GPs, the interaction of which is able to trigger an anti-apoptotic signal conferring a survival advantage to leukemic cells in childhood ALL.

Arenicin-1 (Ar-1) is a β-sheeted antimicrobial peptide from the marine lugworm Arenicola marina . To elucidate the significance of its unique 18-residue cyclic structure and of six cationic arginines for its biological activity and its interaction with biomembranes, we synthesized one linear peptide in which the two cysteines were exchanged for serines (C/S-Ar-1) and a cyclic peptide in which all arginines were replaced by lysines (R/K-Ar-1). We addressed antibacterial and hemolytic activities, the impact of the peptides on bacterial morphology, and their binding to, intercalation into, and permeabilization of model membranes composed of phospholipids or lipopolysaccharide (LPS). In accordance with high salt concentration in sea water, the antibacterial activity of Ar-1 was almost insensitive to high NaCl concentrations. In contrast, the linear derivative lost activity under these conditions against polymyxin B-resistant Proteus mirabilis . Ar-1 intercalated into phospholipid and LPS membranes and formed heterogeneous and short-lived lesions. However, when the peptide was present in both membrane leaflets, it formed defined pores. This characteristic was not observed for the linear derivative C/S-Ar-1. Apparently, the disulfide bond provides conforma-tional stability, which has an impact on salt tolerance, prevents fast degradation by trypsin, and is a prerequisite for the formation of structurally defined pores.

Hepatitis C virus (HCV) infections represent one of the major and still unresolved health problems because current therapy is effective in only 50–80% of cases, depending on viral genotype. A large group of amidinoanthracyclines, with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics, was tested in a high-throughput fluorometric HCV helicase assay. Here, we report the selection of more than 50 potent inhibitors of helicase activity that inhibit the enzyme with IC 50 values in the range of 0.03–10 μ m ; four of these compounds are the most potent inhibitors of helicase activity described in the literature. The activity of these inhibitors is highly dependent on their chemical structure, mainly on the substituent at the amidino carbon atom and on the orientation of the hydroxyl group at the 4′ position of the daunosamine moiety. The most effective compounds act not solely via intercalation into the double-stranded DNA substrate, but also compete with the enzyme for access to the substrate, impeding formation of the active helicase complex. Selected amidinoanthracyclines were tested in the subgenomic HCV replicon system. These experiments confirmed the antiviral activity of two selected inhibitors (EC 50 values below 0.2 μ m with selectivity indices of 19 and 33) and proved that they may be considered as potential anti-HCV drugs.

Circulating neutrophils and monocytes constitute the first line of antibacterial defence, which is responsible for the phagocytosis and killing of microorganisms. Previously, we have described that the staphylococcal cysteine proteinase staphopain B (SspB) cleaves CD11b on peripheral blood phagocytes, inducing the rapid development of features of atypical cell death in protease-treated cells. Here, we report that exposure of phagocytes to SspB critically impairs their antibacterial functions. Specifically, SspB blocks phagocytosis of Staphylococcus aureus by both neutrophils and monocytes, represses their chemotactic activity and induces extensive, nonphlogistic clearance of SspB-treated cells by macrophages. The proteinase also cleaves CD31, a major repulsion (‘do not-eat-me’) signal, on the surface of neutrophils. We suggest that both proteolytic degradation of repulsion signals and induction of ‘eat-me’ signals on the surface of leukocytes are responsible for the observed intensive phagocytosis of SspB-treated neutrophils by human monocyte-derived macrophages. Collectively, this may lead to the depletion of functional neutrophils at the site of infection, thus facilitating staphylococcal colonisation and spreading.

We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides.