Nucleotide-binding oligomerization domain 1 acts in concert with the cholecystokinin receptor agonist, cerulein, to induce IL-33-dependent chronic pancreatitis.

Nucleotide-binding oligomerization domain 1 (NOD1) fulfills important host-defense functions via its responses to a variety of gut pathogens. Recently, however, we showed that in acute pancreatitis caused by administration of cholecystokinin receptor (CCKR) agonist (cerulein) NOD1 also has a role in inflammation via its responses to gut commensal organisms. In the present study, we explored the long-term outcome of such NOD1 responsiveness in a new model of chronic pancreatitis induced by repeated administration of low doses of cerulein in combination with NOD1 ligand. We found that the development of chronic pancreatitis in this model requires intact NOD1 and type I IFN signaling and that such signaling mediates a macrophage-mediated inflammatory response that supports interleukin (IL)-33 production by acinar cells. The IL-33, in turn, has a necessary role in the induction of IL-13 and TGF-ß1, factors causing the fibrotic reaction characteristic of chronic pancreatitis. Interestingly, the Th2 effects of IL-33 were attenuated by the concomitant type I IFN response since the inflammation was marked by clear increases in IFN-γ and TNF-α production but only marginal increases in IL-4 production. These studies establish chronic pancreatitis as an IL-33-dependent inflammation resulting from synergistic interactions between the NOD1 and CCKR signaling pathways.

Expression and production of aberrant PAX5 with deletion of exon 8 in B-lineage acute lymphoblastic leukaemia of children.

Summary We investigated PAX5 expression in childhood B-lineage acute lymphoblastic leukaemia (ALL). Seven of 21 children with B-lineage ALL had multiple PAX5 variants, while 14 children and healthy controls showed full-length (FL) and one variant PAX5. By Western blotting, healthy controls displayed Pax5-FL, while one short Pax5, derived from the deletion of exon 8 (Pax5-DeltaE8) was produced in 90% of ALL samples, as well as in ALL cell lines. PAX5-DeltaE8 lacked more than 50% of the transactivation domain, indicating that aberrant Pax5 production might lead to the arrest of B-cell differentiation, contributing to the pathogenesis of B-lineage ALL.

Age-related changes of alpha-crystallin aggregate in human lens.

Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20-60 S at 37 degrees C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.

dsRNA enhances eotaxin-3 production through interleukin-4 receptor upregulation in airway epithelial cells.

The exacerbation of asthma during viral infections is mainly explained by neutrophils infiltrating into the airways. However, enhanced functions of eosinophils are also observed. The aim of this study was to reveal the mechanism of how eosinophils are activated during and after viral infection of the airways, using a model of viral infection. A synthetic double-stranded RNA, poly inosinic-cytidyric acid (poly(IC)), was transfected to a human airway epithelial cell line (BEAS-2B) and the primary bronchial epithelial cells, to mimic a viral infection. The production of chemokines from the cells was investigated. The transfection of poly(IC), alone, marginally affected the eotaxin-3 production of the cells. However, the transfection of poly(IC) prior to interleukin (IL)-4 stimulation enhanced eotaxin-3 production. Poly(IC) transfection increased mRNA and protein expressions of IL-4 receptor (R)alpha and IL-2Rgamma, components of the IL-4R. In BEAS-2B cells, IL-4-mediated phosphorylation of signal transducer and activator of transcription six was enhanced in poly(IC) transfected cells. This was reversed by the addition of anti-IL-4Ralpha antibody, suggesting the role of an increased number of IL-4 receptors in enhanced IL-4-induced eotaxin-3 production. Poly(IC)-induced upregulation of IL-4Ralpha was inhibited by treatment with cycloheximide or dexamethasone. In conclusion, these results suggest that viral airway infection may enhance interleukin-4-induced eotaxin-3 production through upregulation of the interleukin-4 receptor in airway epithelial cells.

Vasodilative effect of perillaldehyde on isolated rat aorta.

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.

Properties and human origin of two angiotensin-I-converting enzyme inhibitory peptides isolated from a tryptic hydrolysate of human serum albumin.

Two angiotensin-I-converting enzyme (ACE) inhibitory peptides were isolated from a tryptic hydrolysate of human serum albumin (HSA). The peptides were identified by sequencing and other analyses as Ala-Trp and the nonapeptide Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg (human albutensin A), corresponding to f(213-214) and f(210--218) of HSA, respectively. Synthetic versions of both peptides had previously been shown to have ACE inhibitory activity. The present results are the first to show that these peptides have a potential natural origin in humans. Additional studies were done to define the inhibitory properties of these peptides, as they had not been previously reported. The dipeptide and nonapeptide showed dose-dependent inhibition of ACE, with IC50 values of 12 and 1.7 micromol/l, respectively. Lineweaver-Burk plots suggested that Ala-Trp is a competitive inhibitor, and that human albutensin A is a noncompetitive inhibitor.

Isolation of acein-2, a novel angiotensin-I-converting enzyme inhibitory peptide derived from a tryptic hydrolysate of human plasma.

We previously described a novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated Acein-1, that was isolated from a tryptic hydrolysate of human plasma. We now report a second such inhibitory peptide, Acein-2 obtained from the same hydrolysate. The peptide was purified by gel filtration and cation exchange chromatography followed by reversed-phase gradient and isocratic high performance liquid chromatography. Acein-2 was found to be a tripeptide, Leu-Ile-Tyr, which is thought to correspond to f(518-520) of human alpha2-macroglobulin. The synthetic tripeptide showed a potent dose-dependent inhibition of ACE, with an IC(50) value of 0.82 micromol/l. Lineweaver-Burk plots suggested that Acein-2 as well as the previously described Acein-1 are non-competitive inhibitors.

Prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from Japanese patients.

BACKGROUND: Although cagD and cagE (cagDE) identified upstream of cagA have been shown to be involved in the induction of interleukin (IL)-8 expression, the relationship between cagDE status and gastroduodenal diseases still remains to be examined. Thus we investigated prevalence and genetic diversity of cagD, cagE, and vacA in Helicobacter pylori strains isolated from patients with peptic ulcer or gastritis. METHODS: We analyzed 73 H. pylori strains isolated from Japanese patients (gastritis (GA), 15; gastric ulcer (GU), 28; duodenal ulcer (DU), 23; GU and DU, 7). The presence of cagDE was evaluated by polymerase chain reaction (PCR) and Southern hybridization. The vacA genotype was examined by PCR, using type-specific primers. RESULTS: cagDE was present in 13 (86.7%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 21 (91.3%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.89). vacA signal sequence type s1 was found in 14 (93.3%) of 15 patients with GA, 26 (92.9%) of 28 patients with GU, 22 (95.7%) of 23 patients with DU, and 6 (85.7%) of 7 patients with GU and DU (P = 0.84). Sequences of cagDE and vacA in our Japanese strains were highly homologous with one another, and there were no disease-specific mutations. CONCLUSIONS: Most of the H. pylori strains in Japan were cagDE-positive, vacA s1 type, regardless of clinical outcome. The present study also indicated that these genes were conserved well among our H. pylori isolates.

Acein-1, a novel angiotensin-I-converting enzyme inhibitory peptide isolated from tryptic hydrolysate of human plasma.

A novel angiotensin-I-converting enzyme (ACE) inhibitory peptide, designated acein-1, was isolated from the tryptic hydrolysate of human plasma. Gel filtration and cation exchange chromatography were performed to purify this peptide, followed by reversed-phase gradient and isocratic high-performance liquid chromatography. Acein-1 was found to be a heptapeptide, Tyr-Leu-Tyr-Glu-Ile-Ala-Arg, corresponding to f(138-144) of human serum albumin. The synthetic heptapeptide, hexapeptide (Tyr-Leu-Tyr-Glu-Ile-Ala, des-7R acein-1) and octapeptide (Tyr-Leu-Tyr-Glu-Ile-Ala-Arg-Arg, acein-1R) showed dose-dependent inhibitions of ACE, and their IC50 values were 16 micromol/l, 500 micromol/l and 86 micromol/l, respectively. Acein-1 might be a non-competitive inhibitor, while acein-1R may be an uncompetitive inhibitor, as shown by Lineweaver-Burk plots.

Light-induced phase shifting of the circadian conidiation rhythm is inhibited by calmodulin antagonists in Neurospora crassa.

The effects of calmodulin antagonists and inhibitors of protein kinases and phosphatases on light-induced phase shifting were investigated in Neurospora crassa. Calmodulin antagonists, namely, trifluoperazine, chlorpromazine, and W-7, almost completely inhibited the light-induced phase shifting without having any effect on the circadian clock itself. Chlorpromazine was less effective in inhibiting the light-induced phase shifting than trifluoperazine. W-5, a dechlorinated analogue of W-7, failed to inhibit the light-induced phase shifting at the same concentration as that at which W-7 was effective. These results suggest that calmodulin is required during signal transduction from the light-perceiving system to the circadian clock in N. crassa. Inhibitors of protein phosphorylation did not inhibit the light-induced phase shifting, although these inhibitors completely inhibited mycelial growth. Trifluoperazine partially inhibited the phosphorylation of three proteins when phosphorylation was assayed in vitro.

Identification of mutations in DNA polymerase beta mRNAs from patients with Werner syndrome.

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by prematurely aged appearance. Genetic linkage analysis has placed the relevant gene in subchromosomal band 8p12. DNA polymerase beta gene has been mapped to chromosome 8p12-11 and thought to be involved in DNA repair and possibly in recombination. Somatic cells from WS patients exhibit chromosomal instability, a markedly reduced replicative life span and slow growth. The functions of DNA polymerase beta gene and its position prompted us to examine this gene in WS patients. We have found the novel DNA polymerase beta cDNA species in blood samples from WS patients, which contain 107 bp insertions or 87 bp deletions in the catalytic domain of DNA polymerase beta. These mutations change the structure of DNA polymerase beta and thus the capacity of the DNA repair system would be impaired, which may account for the high mutation rate observed in WS.

The MspI restriction fragment length polymorphism of human aldolase B gene on chromosome 9q21.3-q22.2.

The probe containing the exon 9 of human aldolase B gene revealed MspI polymorphism involving two fragments 6.5 and 3.0 kb long with the high frequency of heterozygosity (21%). The two alleles can be distinguished efficiently by the DNA PCR-restriction fragment length polymorphism procedure.

Isolation and analysis of DNA marker revealing restriction fragment length polymorphism from X chromosome specific DNA library.

A human X chromosome specific DNA library was constructed from flow sorted metaphase X chromosomes. Twenty eight single-copy containing phage clones from this X chromosome library were tested for polymorphism against a panel of DNAs from several unrelated individuals, digested with eight restriction enzymes. One (named lambda 33) of these phage clones revealed high frequency two allele polymorphism with the restriction enzyme Msp I and was regionally mapped to the chromosome Xpter by two methods, including Southern analysis with a mapping panel of cell hybrids and quantitative hybridization. The polymorphism appears to be the result of base pair substitutions or modifications rather than DNA rearrangements. In order to examine the heritability of two alleles, the DNAs from four members of a family spanning three generations were examined. The alleles are consistent with their inheritance as a classic X-linked Mendelian locus. Probe lambda 33 will serve as a marker for linkage studies with known polymorphic loci as well as to establish linkage with X-linked diseases.

Quantitative analysis of integrated Ed alpha gene expression in C57BL/6 transgenic mice.

We performed quantitative analysis of Ed alpha gene expression in the transgenic mice, created by microinjecting cloned Ed alpha gene fragments into C57BL/6 fertilized eggs. DNA dot-blot analysis revealed that Ed alpha gene-introduced transgenic mice (B6Ed alpha transgenic mice) contain 20 copies per cell of the Ed alpha gene in their genome. RNA dot-blot analysis revealed that the amount of Ed alpha mRNAs in B6Ed alpha transgenic spleen cells is 20-40-fold higher than those in normal BALB/c or (BALB x C57BL/6)F1 (CBF1) spleen cells. However, the amount of Ed alpha molecules expressed on B6Ed alpha transgenic spleen cells was similar to that expressed on normal BALB/c of CBF1 spleen cells on a gene-dose basis. The amount of endogenous Ed alpha mRNA in the B6Ed alpha transgenic spleen cells was almost equal to that of normal B6 spleen cells. Since the cell surface I-E molecule is formed by non-covalent association of E alpha and E beta chain, these results suggest that, in spite of the high expression of integrated Ed alpha gene in the cytoplasm of B6Ed alpha transgenic mice, the amount of Ed alpha gene expression on the cell surface is limited by the amount of endogenous Eb beta gene products.