Activation of protease-activated receptor 2 stimulates proliferation and interleukin (IL)-6 and IL-8 secretion of endometriotic stromal cells

BACKGROUND: Inflammation has been proposed to play essential roles in the pathophysiology of endometriosis, in which neutrophils and mast cells have been suggested to be involved. We studied whether the protease-activated receptor 2 (PAR2), which is activated by enzymes from neutrophils and mast cells, in endometriotic stromal cells (ESC) has any implication in the development of the disease. METHODS: Cultured ESC were stimulated with various concentrations of a specific PAR2 agonist peptide. Proliferating activity of the cells was determined using immunostaining of proliferating cell nuclear antigen (a cell proliferation marker), 5-bromo-2'-deoxyuridine incorporation into DNA and cell count. The concentrations of interleukin (IL)-6 and IL-8 were measured using specific enzyme-linked immunosorbent assay kits. The phosphorylation of three mitogen-activated protein kinases (MAPK), i.e. p38 MAPK, p42/44 MAPK and stress-activated protein Kinase/c-jun N terminal Kinase, in ESC was examined with Western blot analysis. RESULTS: Activation of PAR2 stimulated the proliferation of ESC and the secretion of IL-6 and IL-8 from ESC in a dose-dependent manner. Activation of PAR2 stimulated the phosphorylation of all three MAPK, and inhibitors of each MAPK suppressed the PAR2 activation-induced proliferation of ESC. CONCLUSIONS: The activation of PAR2 in ESC may be involved in the pathophysiology of endometriosis by inducing the growth and inflammation of endometriotic lesions.     

Regulation of Fungal Infection by a Combination of Amphotericin B and Peptide 2, a Lactoferrin Peptide That Activates Neutrophils

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 × 10⁸ cells) or Aspergillus fumigatus (1 × 10⁸ cells) was prolonged 3 to 5 days by the injection of 10 μg of peptide 2 (a lactoferrin peptide) and 10 μg of α-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 μg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 μg of amphotericin B. When mice received a combined injection of peptide 2 (10 μg/day) with amphotericin B (0.5 μg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as α-defensin 1, β-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O₂⁻) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O₂⁻-generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47^phox as well as p67^phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.     

Polymerization mechanism of N-carboxy-α-amino acid anhydride by organometallic compounds. II.. Reaction with organozinc compounds

Reactions of N-carboxy-α-amino acid anhydride (NCA) with dialkylzinc or related organozinc compounds were studied to elucidate the polymerization mechanism of NCA by dialkylzinc as initiator. The first stage of initiation reaction is a hydrogen abstraction reaction of dialkylzinc from NH group of α-amino acid NCA resulting in the formation of an activated NCA. The second stage of initiation is a reaction between two molecules of the activated NCA forming a zinc carbamate group. Propagation reaction is a carbonyl addition of the zinc carbamate group to the activated NCA to form a mixed anhydride which changes into an amide group releasing carbon dioxide. Regeneration of the activated NCA is supposed to be done by the reaction of free α-amino acid NCA with the zinc atom bonded to nitrogen atom at the growing chain end.     

A nonsynonymous polymorphism in the human fatty acid amide hydrolase gene did not associate with either methamphetamine dependence or schizophrenia

Genetic contributions to the etiology of substance abuse and dependence are topics of major interest. Acute and chronic cannabis use can produce drug-induced psychosis resembling schizophrenia and worsen positive symptoms of schizophrenia. The endocannabinoid system is one of the most important neural signaling pathways implicated in substance abuse and dependence. The fatty acid amide hydrolase (FAAH) is a primary catabolic enzyme of endocannabinoids. To clarify a possible involvement of FAAH in the etiology of methamphetamine dependence/psychosis or schizophrenia, we examined the genetic association of a nonsynonymous polymorphism of the FAAH gene (Pro129Thr) by a case-control study. We found no significant association in allele and genotype frequencies of the polymorphism with either disorder. Because the Pro129Thr polymorphism reduces enzyme instability, it is unlikely that dysfunction of FAAH and enhanced endocannabinoid system induce susceptibility to either methamphetamine dependence/psychosis or schizophrenia.     

Dominant-Negative Rho, Rac, and Cdc42 Facilitate the Invasion Process of Vibrio parahaemolyticus into Caco-2 Cells

To clarify the invasive process of Vibrio parahaemolyticus, an invasion assay was performed using cells expressing dominant negative small GTPases of the Rho family. This assay showed that the dominant negative host phenotype facilitates bacterial invasion, suggesting that the mechanism of V. parahaemolyticus invasion differs from that reported for other invasive bacteria.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

Signal transduction in human B cells initiated via Ig{beta} ligation

Ig and Igß heterodimers are non-covalently associatedwith Ig to compose the antigen receptor complexes on B cells.The demonstration that different sets of tyrosine kinases bindto the cytoplasmic tails of Ig and Igß suggests thatIg and Igß may activate distinct second messengerpathways. In this study, we examined the effects of mAbs againstan exposed epitope of human Igß on pre-B and B celltriggering. Cross-linkage of Igß on B cells leadsto activation of tyrosine kinases, hydrolysis of phosphatidylinositides,and elevation of intracellular Ca²⁺, effects qualitatively identicalto those of anti-_µ mAbs. Our observations thus indicatethat cross-linking of Igß does not segregate signaltransduction pathways connected with the cytoplasmic talls ofIg and Igß. Ig ligation has been reported to be moreeffective in triggering pre-B than B cells, whereas our resultsindicated that Igß ligation is more efficient in triggeringB than pre-B cells. In addition to their activation properties,the anti-Igß mAbs effectively modulated B cell receptorcomplexes and blocked terminal differentiation of all plasmacell isotypes. The findings support the idea that anti-Igßcould serve as a universal B cell immunosuppressant.     

Histological evaluation of medial patellofemoral ligament reconstructed using the Leeds-Keio ligament prosthesis

In a total of 15 knees from 15 patients undergoing medial patellofemoral ligament reconstruction using an artificial substitute and a medial retinaculum slip coverage for recurrent patellar dislocation, the reconstructed ligaments were histologically evaluated using hematoxilin-eosin and elastic van Gieson stains. The artificial substitute was a mesh-type Leeds-Keio ligament. The mean age of the patients at the time of surgery was 20 years (range; 13-38 years). The mean interval from initial reconstruction to gathering was 53 months (range; 11-109 months). In the tissue over the artificial ligament, longitudinally aligned collagen fiber bundles with spindle-shaped nuclei were formed in all specimens except one specimen of 11 months postsurgery, but it seemed to be mature ligament only in specimens more than 60 months postsurgery. The tissue inside the artificial ligament was immature as a whole in all specimens, although 13 out of the 15 specimens had regularly aligned collagen fiber bundles slightly or in some portions.     

Synphilin-1 transgenic mice exhibit mild motor impairments

Synphilin-1 represents a cytoplasmic protein that interacts with α-synuclein and localizes close to synaptic vesicles. The interaction of synphilin-1 with several proteins involved in Parkinson's disease suggests that it might be involved in the pathogenesis of the disease. Nonetheless, the function of synphilin-1 remains unclear. In the present study, we generated transgenic mice expressing human synphilin-1 under the prion protein promoter. Synphilin-1 was widely expressed in neurons in the brain including the substantia nigra, where massive loss of dopamine neurons was not observed. In the transgenic mouse brain, synphilin-1 protein was polyubiquitinated, and partially insoluble. Although modified-SHIRPA revealed no significant difference in behavior and morphology, the reduced rotarod performance and step length were observed in transgenic mice as compared with non-transgenic littermates. Synphilin-1 might be involved in motor function, and its accumulation in the central nervous system can cause motor impairments.     

A new liver metastatic and peritoneal dissemination model established from the same human pancreatic cancer cell line: analysis using cDNA macroarray

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.