Genetic polymorphism of aldehyde oxidase in Donryu rats.

One of major metabolic pathways of [(+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine] (RS-8359), a selective and reversible monoamine oxidase type A inhibitor, is the aldehyde oxidase-catalyzed 2-hydroxylation at the pyrimidine ring. Donryu rats showed a dimorphic pattern for the 2-oxidation activity with about 20- to 40-fold variations in the Vmax/Km values between a low and a high activity group. The rats were classified as extensive metabolizers (EM) and poor metabolizers (PM) of RS-8359, of which ratios were approximately 1:1. One rat among the EM rats of each sex showed extremely high activity, and they were referred to as ultrarapid metabolizers. There was no significant difference in the expression levels of mRNA of aldehyde oxidase between the EM and PM rats. Analysis of nucleotide sequences showed four substitutions, of which the substitutions at 377G>A and 2604C>T caused 110Gly-Ser and 852Ala-Val amino acid changes, respectively. Amino acid residue 110 is located very near the second Fe-S center of aldehyde oxidase. Its change from nonchiral Gly to chiral Ser may result in a conformational change of aldehyde oxidase protein with the shift of isoelectric point value from 5.0 in the EM rats to 6.2 in the PM rats. The 110Gly-Ser amino acid substitution (377G>A) may be primarily responsible for the variations of aldehyde oxidase activity observed in Donryu rats, in addition to the difference of expression levels of aldehyde oxidase protein. If a new drug candidate is primarily metabolized by aldehyde oxidase, attention should be given to using a rat strain with high aldehyde oxidase activity and small individual variation.     

Positive interactions between human interferon and cepharanthin against human cancer cells in vitro and in vivo

A human tumor microcytotoxicity-viable cell-staining assay was used to test the antiproliferative effect of recombinant human interferon-beta or-gamma alone and in combination with bisbenzylisoquinoline alkaloid cepharanthin against four human tumor cell lines in vitro and in nude mice. Results obtained in the in vitro study indicate that combinations of interferon-beta/gamma with cepharanthin show synergistic and, occasionally, additive antiproliferative effects in a dose-dependent manner on tumor viable cell-staining assay. Interferon-gamma combined with cepharanthin suppressed the growth of all four human tumor cell lines (RPMI 4788, PC 10, HeLa, ZR-75-1), and this enhanced antiproliferative effect was not dependent on the interferon species involved, including interferon-beta and-gamma. In an experimental model of pulmonary metastasis, in which human colon tumor cells were inoculated i.v. into nude mice, interferon-gamma alone exerted significant inhibitory activity against pulmonary metastasis in a dose-dependent manner, and cepharanthin alone also significantly inhibited metastasis. Furthermore, a combination of interferon-gamma with cepharanthin resulted in a considerable suppression of pulmonary metastasis. These studies indicate that due to their therapeutic potential, combinations of recombinant human interferon-beta or-gamma with cepharanthin might be a promising therapy for pulmonary metastasis of human cancers.     

Modulation of fibroblast proliferation and transformation by activated macrophages during postoperative peritoneal reepithelialization

We studied the modulation of fibroblast proliferation and transformation by postoperative macrophages. One group of rabbits underwent resection and reanastomosis of the small bowel, after which macrophages were collected by peritoneal lavage. A second group of rabbits underwent peritoneal wall abrasion followed by collection of local fibroblast on postoperative days 4 and 8. Postoperative macrophages were added to five culture dishes containing fibroblasts. After 24 hours, tritiated thymidine was added to the culture dishes and incubated overnight. In two other dishes, which were incubated for up to 8 days, a 24-hour pulse of tritiated thymidine was added before culture termination. Postoperative day 4 fibroblasts demonstrated a greater increase in cell number during the culture interval compared to fibroblasts collected on postoperative day 8. By the second day of coculture with macrophages collected from different postoperative days, tritiated thymidine incorporation by day 4 fibroblasts was suppressed, especially by postoperative day 7 macrophages. Thereafter, a stimulation in tritiated thymidine uptake was found. In contrast, tritiated thymidine uptake by day 8 fibroblasts was accelerated by coculture with macrophages, especially those collected on postoperative day 7. Day 4 fibroblasts assumed a more spindly appearance when cocultured with macrophages than did day 8 fibroblasts. Taken together, these data suggest that macrophages activated in response to surgical injury may secrete substances that induce proliferation and transformation of fibroblasts.     

Macrophage migration inhibitory factor and the discovery of tautomerase inhibitors

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine released from T-cells and macrophages, and is a key molecule in inflammation. Although a detailed understanding of the biological functions of MIF has not yet been found, it is known that MIF catalyzes the tautomerization of phenylpyruvate and a non-physiological molecule, D-dopachrome. A potent tautomerase inhibitor would be expected, as a validation tool, to shed light on role of MIF activity and the relationship between its biological and enzymatic activity. Such tautomerase inhibitors would be useful in the treatment of MIF-related diseases, such as sepsis, acute respiratory distress syndrome (ARDS), asthma, atopic dermatitis, rheumatoid arthritis (RA), nephropathy and tumors. In this review, we have focused on (1) the biological and enzymatic activities of MIF, (2) the discovery of novel, drug-like tautomerase inhibitors of MIF using a structure-based computer-assisted search, and (3) a crystallographic and molecular modeling study of the MIF-tautomerase inhibitor complexes (A review with 133 references).     

Progenitor analysis of primitive erythropoiesis generated from in vitro culture of embryonic stem cells

OBJECTIVE: A variety of hematopoietic lineage cells have been produced from embryonic stem (ES) cells, but their differentiation processes have not been elucidated well, especially from the point of view of progenitor analysis. In this study, we utilized our coculture system, in which ES-derived Flk-1+ cells differentiated into TER-119+ primitive erythroid (EryP) cells on OP9 cells, and looked for progenitors in primitive erythropoiesis. MATERIALS AND METHODS: We studied the kinetics of TER-119+ erythroblast generation from Flk-1+ cells by monitoring the expression of TER-119, CD41, VE-cadherin, CD34, and c-kit antigens. Multicolor analysis was performed to detect CD41+TER-119+ cells and the stained cells were sorted to examine their morphology and EryP-producing potential in colony formation. RESULTS: Kinetic studies showed that the CD41+ population appeared early in the coculture and its expression pattern implied a role as an immediate progenitor of TER-119+ EryP cells. Multicolor analysis and colony-formation study supported this notion. Other progenitor markers such as VE-cadherin, CD34, and c-kit did not seem to define an immediate progenitor of EryP cells. One interesting observation is the detection of unique populations, CD41dim and CD41bright, detectable after 48 hours of the coculture. Majority of the CD41dim population progressed to the EryP lineage, whereas the CD41bright population seemingly advanced on a pathway distinct from the CD41dim population. CONCLUSIONS: CD41 expression was a useful marker to trace hematopoietic progenitors in ES-derived differentiation system. In particular, the CD41dim but not CD41bright population could serve as immediate precursors of EryP cells.     

Inhaled nitric oxide attenuates apoptosis in ischemia-reperfusion injury of the rabbit lung

Background

Lung ischemia-reperfusion injury occurs after lung transplantation and various clinical procedures. Recently, apoptosis was reported to be induced after ischemia-reperfusion. We investigated the effects of inhaled nitric oxide (NO) on lung ischemia-reperfusion and apoptosis after ischemia-reperfusion.

Methods

As a control group, the left pulmonary hilum of Japanese white rabbits (n = 10) was occluded for 120 minutes and reperfused for 120 minutes. In the inhaled NO group (n = 10), 20 parts per million nitric oxide was inhaled during reperfusion. The sham-operated group was ligated at the right hilum and perfused by the left lung only for 120 minutes. The mean pulmonary arterial pressures and Pao₂ were measured during reperfusion. The wet-to-dry weight ratio of the left lower lobe of the lung was calculated. The number of apoptotic cells was estimated using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) technique. The TUNEL staining for a time course study was done using 15 control animals that were killed by exsanguination at 15, 30, and 60 minutes after reperfusion.

Results

After 120 minutes of reperfusion, the mean pulmonary arterial pressures in the control group and in the inhaled NO group were 23.0 ± 3.2 mm Hg and 13.6 ± 2.4 mm Hg, respectively (p < 0.01). At the same time point, the Pao₂ in the control group and in the inhaled NO group were 46.1 ± 15.9 mm Hg and 88.1 ± 14.7 mm Hg, respectively (p < 0.01). The wet-to-dry weight ratios in the control group and in the inhaled NO group were 0.856 ± 0.024 and 0.808 ± 0.006, respectively (p < 0.01). Apoptotic cells appeared in the early phase of reperfusion (after 15 minutes' reperfusion). The number of apoptotic cells was significantly lower in the inhaled group than in the control group after 120 minutes' reperfusion (1.76% versus 2.87%, p < 0.01).

Conclusions

Our results suggest that the inhaled NO prevents lung ischemia-reperfusion injury and attenuates apoptosis after reperfusion in the rabbit lung.     

Induction of CD28 on the new myeloma cell line MOLP-8 with t(11;14)(q13;q32) expressing delta/lambda type immunoglobulin

The novel multiple myeloma (MM) cell line MOLP-8 carrying the t(11;14) (q13;q32) was established from the peripheral blood of a 52-year-old Japanese male patient with Bence-Jones delta/lambda type MM (stage IIIA with hyperammonemia). The growth of MOLP-8 cells is constitutively independent of exogenous growth factors or feeder cells. MOLP-8 cells grow mainly as free floating single cells and slightly adherent on the bottom of the plastic culture flask. Wright-Giemsa-stained MOLP-8 cells show the typical plasma cell morphology with abundant cytoplasm, heterogeneous cell size and one to three nuclei. The immunoprofile of MOLP-8 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) delta/lambda chains, CD10, CD29, CD38, CD40, CD44, CD49b, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. CD28 became positive after co-culture of MOLP-8 cells with bone marrow adherent stromal (BST) feeder cells for a week. About 30% of MOLP-8 cells adhered strongly to the BST cells, but the cellular adhesion was clearly inhibited by addition of either anti-CD29 or anti-CD106 monoclonal antibody, suggesting a specific cellular adhesion through alpha4beta1-integrin-VCAM-1 interaction. The novel MOLP-8 cell line together with the present myeloma cell lines will present useful model systems in the investigation of the biology of MM.