Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells.

Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE+PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis.

Polyethyleneimine renders mitochondrial membranes permeable by interacting with negatively charged phospholipids in them.

Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization.

Transmission of External Environmental pH Information to the Inside of Liposomes via Pore-Forming Proteins Embedded within the Liposomal Membrane.

Liposomes are closed-membrane vesicles comprised of lipid bilayers, in which the inside of the vesicles is isolated from the external environment. Liposomes are therefore often used as models for biomembranes and as drug delivery carriers. However, materials encapsulated within liposomes often cannot respond to changes in the external environment. The ability of enclosed materials to maintain their responsiveness to changes in the external environment following encapsulation into liposomes would greatly expand the applicability of such systems. We hypothesize that embedding pore-like "access points" into the liposomal membrane could allow for the transmission of information between the internal and external liposomal environments and thus overcome this inherent limitation of conventional liposomes. To investigate this, we evaluated whether a change in the pH of an external solution could be transmitted to the inside of liposomes through the pore-forming protein, yeast voltage-dependent anion channel (VDAC). Transmission of a pH change via VDAC was evaluated using a polyglutamic acid/doxorubicin complex (PGA/Dox) as an internal pH sensor. Upon encapsulation into conventional liposomes, PGA/Dox exhibits no pH sensitivity due to isolation from the external environment. On the other hand, PGA/Dox was found to retain its pH sensitivity upon encapsulation into VDAC-reconstituted liposomes, suggesting that VDAC facilitated the transmission of information on the pH of the external environment to the inside of the liposomes. In conclusion, we successfully demonstrated the transmission of information between the external and internal liposomal environments by a stable pore-like structure embedded into the liposomal membranes, which serve as access points.

Specific formation of trypsin-resistant micelles on a hydrophobic peptide observed with Triton X-100 but not with octylglucoside.

The manner of interaction of the coat peptide of the Pf3 phage (Pf3 peptide) with lipid bilayers has been extensively studied. Presently, we designed a derivative of the Pf3 peptide, referred to as the DDRK peptide, and subjected it to trypsin digestion to understand its physicochemical properties. In the presence of Triton X-100 used for solubilization of the peptide, digestion of DDRK with trypsin caused specific cleavage at the lysine (Lys) residue in its N-terminal region but not at other Lys residues or at the arginine residue. As the N-terminal region of the DDRK peptide is relatively hydrophilic, but its remaining region is hydrophobic, this hydrophobic region of the peptide would be expected to be coated by Triton micelles. Thus, we propose that the presence of such micelles protected against cleavage there, leading to selective cleavage by trypsin of the DDRK peptide at its hydrophilic Lys residue in the N-terminal part of the molecule. However, such a protective effect on the DDRK peptide against trypsin digestion was not observed with octylglucoside. The observed results are important for better understanding of the manner of interaction between detergents and hydrophobic peptides.

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

Substitution of certain amino acids in a short peptide causes a significant difference in their immunoreactivities with antibodies against different epitopes: evidence for possible folding of the peptide on a nitrocellulose or PVDF membrane.

Substitution of amino acids in a peptide caused remarkable differences in its immunoreactivities with antibodies against 3 epitopes in the immobilized peptide. The observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.

Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques.

Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100.

Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin.

To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl-2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mitochondrial configurations or by measurement of the permeability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release.