Kiyohiro Irimajiri

Protease-activated receptor-2 (PAR-2) in the rat gastric mucosa: immunolocalization and facilitation of pepsin/pepsinogen secretion

Agonists of protease‐activated receptor‐2 (PAR‐2) trigger neurally mediated mucus secretion accompanied by mucosal cytoprotection in the stomach. The present study immunolocalized PAR‐2 in the rat gastric mucosa and examined if PAR‐2 could modulate pepsin/pepsinogen secretion in rats. PAR‐2‐like immunoreactivity was abundant in the deep regions of gastric mucosa, especially in chief cells. The PAR‐2 agonist SLIGRL‐NH2, but not the control peptide LSIGRL‐NH2, administered i.v. repeatedly at 0.3 – 1 μmol kg−1, four times in total, significantly facilitated gastric pepsin secretion, although a single dose produced no significant effect. The PAR‐2‐mediated gastric pepsin secretion was resistant to omeprazole, NG‐nitro‐L‐arginine methyl ester (L‐NAME) or atropine, and also to ablation of sensory neurons by capsaicin. Our study thus provides novel evidence that PAR‐2 is localized in mucosal chief cells and facilitates gastric pepsin secretion in the rats, most probably by a direct mechanism.

Clonal origin of Philadelphia chromosome negative cells with trisomy 8 appearing during the course of α-interferon therapy for Ph positive chronic myelocytic leukemia

The transient appearance of a Philadelphia chromosome (Ph) negative clone with trisomy 8 was found in the bone marrow cells from a patient with Ph positive CML during the course of alpha-interferon (IFN) therapy. To determine whether this clone was derived from a Ph positive clone or from some other cell lineage, we performed molecular cytogenetic studies on bone marrow cells from the patient by fluorescence in situ hybridization (FISH). No fusion of BCR-ABL could be detected in cells with trisomy 8, clearly indicating that the Ph negative trisomy 8 clone was not derived from the Ph positive CML population.

Induction of apoptosis in HL-60 cells treated with medicinal herbs

In order to develop a new apoptosis inducer, we screened 22 crude drugs for their apoptosis-inducing activity. It was found that Glycyrrhiza uralensis, Cynomorium songaricum, Eucommia ulmoides, Phellodendron amurense, Cinnamomum cassia and Paeonia lactiflora induced the death of HL-60 cells. To investigate the mechanism of apoptosis induced by these six crude drugs, the mitochondrial transmembrane potential and the activity of caspase-3 were measured. Reduced mitochondrial transmembrane potentials within 12 hours after the administration of Glycyrrhiza uralensis, Cynomorium songaricum, Phellodendron amurense and Paeonia lactiflora, and within 24 hours after the administration of Eucommia ulmoides and Cinnamomum cassia were observed. All of the six apoptosis-inducing crude drugs increased caspase-3 activity within 12-36 hours after administration. After further examining the apoptosis-inducing activity of berberine, palmatine, panelofuroline and glycyrrhizin, which were the ingredients obtained from Phellodendron amurense, Glycyrrhiza uralensis and Paeonia lactiflora, it was found that only berberine could induce apoptosis. From these results, it was concluded that the apoptosis induced by the six crude drugs (Glycyrrhiza uralensis, Cynomorium songaricum, Eucommia ulmoides, Phellodendron amurense, Cinnamomum cassia and Paeonia lactiflora) occurred via the mitochondrial route and that the apoptosis-conducting mechanism acted through a cascade involving caspase-3.

A new bisphosphonate, YM529 induces apoptosis in HL60 cells by decreasing phosphorylation of single survival signal ERK

It is believed that bisphosphonates (BPs) induce apoptosis in cells such as myeloma cells, as they inhibit prenylation of G-proteins. However, the details of the apoptosis-inducing mechanism remain obscure. In the present study, we attempted to clarify the mechanism by which YM529, a new bisphosphonate, induces apoptosis. YM529 induced cell deaths in HL60 cells in a concentration-dependent manner. At that time, we observed an increase in Caspase-3 activity and morphological fragmentation of the nuclei. We could confirm that these cell deaths were evidence of apoptosis. The apoptosis induced by YM529 was not inhibited by the addition of farnesyl pyrophosphate (FPP), but was by the addition of geranylgeranyl pyrophosphate (GGPP). When we examined the survival signals at the time of apoptotic induction, we also observed that the administration of YM529 caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). However, other survival signals such as nuclear factor kappa B (NF-kappaB), protein kinase B (Akt), and p38 mitogen-activated protein kinase (p38) exhibited no change. In addition, no quantitative change was observed in Bcl-2, which is an anti-apoptosis protein. It was also observed that apoptosis was induced when U0126, an MEK inhibitor, was added to the cells to inhibit ERK. These results suggest that YM529, the new bisphosphonate, induced apoptosis when inhibit GGPP synthase and consequently decreased the levels of phosphorylated ERK, which is a survival signal; moreover, during this process, there is no influence on NF-kappaB, Akt, p38, and Bcl-2. The results of this study also suggest that YM529 can be used as an anticancer agent, in addition to its use as a therapeutic agent to treat osteoporosis.

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma.

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.

Inhibition of growth and induction of apoptosis by all‐trans retinoic acid in lymphoid cell lines transfected with the PML/RARα fusion gene

The interaction of an exogenous PML/RARα fusion gene associated with acute promyelocytic leukaemia, with all‐trans retinoic acid (ATRA) was examined in two lymphoid cell lines. L1210 and MOLT‐4 cells were transfected with PML/RARα cDNA in the expression vector pGD and stable transformants (L1210PML/RARα and MOLT‐4PML/RARα) were selected with G418. ATRA inhibited the growth of these stable transformants, as assessed by [3H]thymidine incorporation, in a dose‐dependent manner, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also induced apoptosis, as assessed by fragmentation of genomic DNA, in L1210PML/RARα and MOLT‐4PML/RARα cells but not in control cells. The exogenous PML/RARα fusion gene therefore probably mediates the effects of ATRA on cell growth and apoptosis in these cell lines.

CD3 down‐regulating factor in sera and culture supernatants of leukaemic cells from patients with adult T cell leukaemia

Immunological abnormality of T lymphocytes in patients with adult T cell leukaemia (ATL) is characterized by abnormal expression of the 55 kD chain of the receptor for interleukin 2 (IL‐2R/p55) (Tac), and the down‐regulation of CD 3 expression. Using serum and culture supernatants of leukaemic cells from ATL patients (Group A) whose CD 3 expression was down‐regulated and those (Group B) whose CD 3 was not low, the possible mechanism of CD 3 down‐regulation on ATL cells was discussed. When PBMC from normal individuals were cultured with sera from ATL patients for 24 h, CD 3 expression revealed by mean fluorescent intensity (MFI) was down‐regulated by sera from ATL patients in Group A (MFI: Pt 1 = 51.6 ± 4.5, Pt 2 = 48.0 ± 6.9, control = 96.5 ± 6.6), not by sera from patients in Group B (MFI: Pt 3 = 105.5 ± 7.9, Pt 4 = 102.5 ± 8.3, control = 96.5 ± 6.6). When normal PBMC were cultured with supernatants of leukaemic cells from ATL patients in Group A, this CD 3 down‐regulating activity was also detected (MFI: Pt 1 = 78.0 ± 10.2, Pt 2 = 70.6 ± 8.7, control = 94.0 ± 6.6). By using gel‐chromatography, the fractionated supernatants from ATL patients in Group A decreased CD 3 expression of normal PBMC significantly (MFI: Pt 1 = 22.9 ± 5.8, Pt 2 = 28.8 ± 7.4, control = 92.1 ± 9.6). This CD 3 down‐regulating activity in fractionated supernatant was not inhibited by any lymphokine antibodies, anti‐IL‐1α antibody (Ab), anti‐IL‐1B Ab, anti‐IL‐2 Ab, anti‐IL‐3 Ab, anti‐IL‐4 Ab, anti‐IL‐6 Ab, anti‐TNF‐α Ab and anti‐IFN‐γ Ab. Any known cytokines (IL‐1, IL‐2, IL‐3, IL‐4, IL‐6, TNF‐α and IFN‐γ) could not modulate CD 3 expression of normal PBMC. These findings suggested that there are novel factor(s) with CD 3 down‐regulating activity in the serum and culture supernatant of ATL patient and those factor(s) are involved in progression of ATL.

Occurrence of heavy chain of 7S IgM half-molecule whose NH2-terminal sequence is identical with that of κ light chain sequence in patients with Waldenstrom macroglobulinemia

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique mu heavy chain and kappa light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1-20 were identical to those of the NH2-terminal region of kappa light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique mu chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known mu chain to residue 19 in the known CH1 domain and a sequence spanning from residues 67-82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a kappa light chain and a unique mu heavy chain of 35 kDa polypeptide in which the NH2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of kappa light chain in the NH2-terminal region.

Establishment of a myelodysplastic syndrome (MDS)/secondary AML-derived T lymphoid cell line K2-MDS

We have established a T lymphoid cell line, K2-MDS, from the peripheral blood mononuclear cells (PBMC) of a patient with acute myeloblastic leukemia (AML) transformed myelodysplastic syndrome (MDS). K2-MDS cells are positive for the expression of CD4, CD5, CD13, CD25, CD71, CD95, HLA-DR and cytoplasmic CD3. Southern blotting analysis shows T cell receptor (TCR) beta chain genes rearrangements, whereas immunoglobulin heavy chain (IgH) genes are not rearranged. Further, the patient PBMC contains TCR beta chain genes rearrangements in the same manner as K2-MDS cells. The data indicate that K2-MDS is a T lymphoid cell line derived from a myelodysplastic clone in the patient PBMC. This new MDS-derived cell line K2-MDS may be a useful in vitro model for studies on the pathogenetic mechanisms leading to MDS.

Elevated levels of soluble ICAM-1 in serum of patients with acute myeloid leukemia undergoing bone marrow transplantation

Serum soluble ICAM‐1 concentrations were measured in 10 patients with or without chronic graft‐vs.‐host disease (GVHD) after allogeneic bone marrow transplantation. The serum soluble ICAM‐1 levels in the patients with chronic GVHD were significantly higher than that in the patients without chronic GVHD. The data indicated that serum soluble ICAM‐1 is a useful parameter for predicting chronic GVHD.