No abstract

The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Singlet oxygen ( 1 O 2 ) is a reactive oxygen species that may be generated in biological systems. Photodynamic therapy generates 1 O 2 by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. 1 O 2 oxidizes amino acid side chains of proteins and inactivates enzymes when generated in vitro. Among proteogenic amino acids, His, Tyr, Met, Cys, and Trp are known to be oxidized by 1 O 2 at physiological pH. However, there is a lack of direct evidence of oxidation of proteins by 1 O 2 . Because 1 O 2 is difficult to detect in cells, identifying oxidized cellular products uniquely derived from 1 O 2 could serve as a marker of its presence. In the present study, 1 O 2 reactions with model peptides analyzed by tandem mass spectrometry provide insight into the mass of prominent adducts formed with the reactive amino acids. Analysis by MALDI-TOF and tandem mass spectrometry of peptides of cytochrome c exposed to 1 O 2 generated by photoexcitation of the phthalocyanine Pc 4 showed unique oxidation products, which might be used as markers of the presence of 1 O 2 in the mitochondrial intermembrane space. Differences in the elemental composition of the oxidized amino acid residues observed with cytochrome c and the model peptides suggest the protein environment can affect the oxidation pathway.

Abstract10-N-nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipindeficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

Factors such as charge, aggregation and lipophilicity influence photosensitiser localisation. The lipophilic octasubstituted sensitiser 2,3,9,10,16,17,23,24-octakis(decyloxy)phthalocyaninato zinc(II) was incorporated into liposomes of dimyristoyl-L-alpha-phosphatidylcholine (DMPC), non-ionic micelles of Tween 80 and the hydrosoluble polymer Solutol HS 15 in order to investigate how these different environments affect the photophysical properties and phototoxicity of the photosensitiser. Fluorescence quantum yields and singlet molecular oxygen generation are enhanced in the presence of Solutol HS 15. Phototoxicities were calculated by employing a concentration of 10(-7) M of the dye against the Hep-2 cell line, which showed a viability of 53 and 30% in DMPC and Solutol HS 15, respectively. After 24 h of photodynamic therapy with 15 min irradiation, apoptotic and necrotic cells were observed.

SUMMARY We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4+ T cells and up-regulate TCR triggered- IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4+ T cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4+ T cells express TLR2 and respond to TLR2 co-stimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T cell TLR2 in priming of CD4+ T cells in vivo, naïve MTB Ag85B specific TCR transgenic CD4+ T cells (P25 TCR-Tg) were adoptively transferred into Tlr2-/- recipient mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3Cys-SKKKK (P3CSK4). TLR2 engagement during priming resulted in increased numbers of IFN-γ secreting P25 TCR-Tg T cells one week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4+ T cells increases MTB-Ag specific responses and may contribute to protection against MTB infection.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40 ϩ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.