The Puzzling Problem of Cardiolipin Membrane-Cytochrome c Interactions: A Combined Infrared and Fluorescence Study.

The interaction of cytochrome c (cyt c) with natural and synthetic membranes is known to be a complex phenomenon, involving both protein and lipid conformational changes. In this paper, we combined infrared and fluorescence spectroscopy to study the structural transformation occurring to the lipid network of cardiolipin-containing large unilamellar vesicles (LUVs). The data, collected at increasing protein/lipid ratio, demonstrate the existence of a multi-phase process, which is characterized by: (i) the interaction of cyt c with the lipid polar heads; (ii) the lipid anchorage of the protein on the membrane surface; and (iii) a long-distance order/disorder transition of the cardiolipin acyl chains. Such effects have been quantitatively interpreted introducing specific order parameters and discussed in the frame of the models on cyt c activity reported in literature.

The key role played by charge in the interaction of cytochrome c with cardiolipin.

Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c-CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys â†’ Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein.

Omega-3 and Omega-6 Fatty Acids Act as Inhibitors of the Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 Activity.

Polyunsaturated fatty acids have been reported to play a protective role in a wide range of diseases characterized by an increased metalloproteinases (MMPs) activity. The recent finding that omega-3 and omega-6 fatty acids exert an anti-inflammatory effect in periodontal diseases has stimulated the present study, designed to determine whether such properties derive from a direct inhibitory action of these compounds on the activity of MMPs. To this issue, we investigated the effect exerted by omega-3 and omega-6 fatty acids on the activity of MMP-2 and MMP-9, two enzymes that actively participate to the destruction of the organic matrix of dentin following demineralization operated by bacteria acids. Data obtained (both in vitro and on ex-vivo teeth) reveal that omega-3 and omega-6 fatty acids inhibit the proteolytic activity of MMP-2 and MMP-9, two enzymes present in dentin. This observation is of interest since it assigns to these compounds a key role as MMPs inhibitors, and stimulates further study to better define their therapeutic potentialities in carious decay.

Salivary miRNAome profiling uncovers epithelial and proliferative miRNAs with differential expression across dentition stages.

Saliva's ability to mirror the internal physiological environment of an organism coupled with its facile accessibility makes it an attractive diagnostic medium. The finding of microRNAs (miRNAs) in saliva has expanded the field of biomarker discovery since these tiny non-coding RNAs affect various physiological processes and diseases. Few reports have linked miRNAs to tooth development and eruption, with none having studied this in humans. As a first initiative to describe miRNAs in saliva whose modulations may reflect developing and erupting teeth, we quantified the levels of 730 miRNAs in the saliva of children of varying dentition stages: edentulous (newborns), deciduous and permanent by megaplex stemloop reverse-transcription quantitative PCR. The three groups expressed 193, 181 and 192 miRNAs, respectively, where 125 miRNAs had consistent expression. The remaining miRNAs had inter-group variations from 5 to hundreds of fold, where most had either an increasing or decreasing trend in going from edentulous to deciduous to permanent. A literature survey of epithelial miRNAs found most were present in saliva. Moreover, many miRNAs with expression differences between groups had previously documented functions in proliferation, cell cycle, apoptosis and other cellular behaviours key to the dynamics of tooth morphogenesis. Lastly, miRNAs of the same family, such as the let-7 and miR-200 families, or transcribed from the same hairpin, had similar expression patterns. The results presented here should serve as a salivary miRNA dictionary for future studies in tooth development as well as in childhood diseases associated with modulations in saliva composition.