ABSTRACT
Secretagogin is a six EF-hand calcium-binding protein that can identify granule cells in the dentate gyrus of hippocampus. The aim of this study was to determine if secretagogin can be detected in human blood cells. Eight adult males were recruited for blood analysis. Whole blood was separated into plasma, peripheral mononuclear cells and erythrocytes with Ficoll-Paque and probed for secretagogin using reverse-transcription polymerase chain reaction and Western blot. While secretagogin mRNA was detected in both peripheral mononuclear cells and erythrocytes using reverse-transcription polymerase chain reaction, SCGN protein was only detected in erythrocytes. Interestingly, peripheral mononuclear cells secretagogin mRNA expression levels showed significant negative correlation with age. This begets the question on the function of secretagogin in blood cells and if it is correlated to neurodegeneration associated with ageing. This remains our impetus for further research.
Subject(s)
Calcium-Binding Proteins/metabolism , Erythrocytes/metabolism , Adult , Age Factors , Blotting, Western , Calcium-Binding Proteins/genetics , Humans , Male , Neutrophils/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Secretagogins , Young AdultABSTRACT
We evaluated the therapeutic effect of secretory phospholipase A2 (sPLA2)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA2 inhibition.
Subject(s)
Peptides/pharmacology , Phospholipases A/antagonists & inhibitors , Time , Animals , Cartilage, Articular/drug effects , Cartilage, Articular/pathology , Cartilage, Articular/ultrastructure , Cell Line , Cell Survival/drug effects , Disease Models, Animal , Inflammation , Macrophages/drug effects , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Microscopy, Electron, Transmission/methods , Osteochondritis/pathology , Osteochondritis/prevention & control , Phospholipases A2 , Synovitis/pathology , Synovitis/prevention & control , Tarsal Joints/drug effects , Tarsal Joints/pathology , Tarsal Joints/ultrastructure , Tumor Necrosis FactorsABSTRACT
The functional site of 'phospholipase A2 inhibitor from python' (PIP) was predicted based on the hypothesis of proline brackets. Using different sources of secretory phospholipase A2 (sPLA2s) as enzyme, and [3H]arachidonate-labelled Escherichia coli as substrate, short synthetic peptides representing the proposed site were examined for their secretory phospholipase A2 (sPLA2) inhibitory activity. A decapeptide P-PB.III proved to be the most potent of the tested peptides in inhibiting sPLA2 enzymatic activity in vitro, and exhibited striking anti-inflammatory effects in vivo in a mouse paw oedema model. P-PB.III inhibited the enzymatic activity of class I, II and III PLA2s, including that of human synovial fluid from arthritis patients. When tested by ELISA, biotinylated P-PB.III interacted positively with various PLA2s, suggesting that the specific region of PIP corresponding to P-PB.III, is likely to be involved in the PLA2-PLI interaction. The effect of P-PB.III on the peritoneal inflammatory response after surgical trauma in rats was also examined. P-PB.III effectively reduced the extent of postsurgical peritoneal adhesions as compared to controls. sPLA2 levels at seventh postoperative day in the peritoneal tissue of P-PB.III-treated rats were also significantly reduced (P < 0.05) in comparison to those of the untreated controls. The present results shed additional insight on the essential structural elements for PLA2 binding, and may be useful as a basis for the design of novel therapeutic agents.
