Kunihiko Itoh

Relationship of urinary pseudouridine and 1-methyladenosine to activity of leukemia and lymphoma

Urinary levels of pseudouridine and 1-methyladenosine in patients with leukemia and lymphoma were measured by the inhibition ELISA using monoclonal antibodies to determine the correlation of nucleosides excretion with disease activity. Significantly elevated levels of these nucleosides were detected in patients with all types of disease tested. Seventy-seven percent (46/60) and 62% (37/62) of patients had elevated levels of pseudouridine and 1-methyladenosine above normal mean + 2S.D., respectively, and combination assay of these nucleosides gave higher positive rate (87%; 52/60) than either single assay. The changes of urinary pseudouridine and 1-methyladenosine reflected the disease status of patients in remission or in relapse and the effect of chemotherapy. These results suggest that urinary pseudouridine and 1-methyladenosine might be clinically useful as complementary markers to the monitoring of the disease status of patients with leukemia and lymphoma by hematological examination.

An immunohistochemical analysis for cancer of the esophagus using monoclonal antibodies specific for modified nucleosides

Background. Modified nucleosides such as 1‐methyladenosine and pseudouridine exist as minute components of transfer ribonucleic acid (tRNA) and are excreted in the urine in large amounts in the presence of malignancy. Although use of these modified nucleosides as tumor markers has long been studied and many reports have detailed their relationship with malignant tumors and the urinary excretion of various modified nucleosides, there have been no reports on modified nucleosides in esophageal carcinoma.

Detection of elevated amounts of urinary pseudouridine in cancer patients by use of a monoclonal antibody

Preparation of anti-pseudouridine monoclonal antibodies (MoAbs) and their applications for the quantitation of urinary pseudouridine in cancer patients are described. Seven MoAbs were selected. Five MoAbs were specific for pseudouridine and two MoAbs were cross-reactive with uridine. The most specific antibody, APU-6, was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary pseudouridine. Sensitivity was in the picomole range and the accuracy was nearly equal to that of the high performance liquid chromatography (HPLC) assay. The amount of pseudouridine in the urine of 28 healthy donors was 31.17 +/- 9.94 nmol/mumol creatinine. In 55% (35/63) of patients with cancer, urinary pseudouridine was elevated above the normal mean + 2 SD (51.04 nmol/mumol creatinine). Particularly, all of the patients (15/15) with leukemia and lymphoma had elevated levels of pseudouridine. These results suggest that urinary pseudouridine might be useful as a marker for leukemia and lymphoma.

Preparation of a Monoclonal Antibody Specific for 1‐Methyladenosine and Its Application for the Detection of Elevated Levels of 1‐Methyladenosine in Urines from Cancer Patients

A monoclonal antibody specific for a modified nucleoside, 1‐methyladenosine, was prepared and characterized. This antibody, termed AMA‐2, reacts with 1‐methyladenosine and 1‐methyladenine but not with other nucleosides, particularly methylated adenosines other than 1‐methyladenosine and methylated guanosines, tested in this investigation. In our experiments, AMA‐2 was used in an enzyme‐linked immunosorbent assay (ELISA) system for the quantitation of the levels of 1‐methyladenosine in urine. Sensitivity was in the picomole range and accuracy was nearly equal to that of the high‐performance liquid chromatography (HPLC) assay system. Urinary levels of 1‐methyladenosine in healthy donors and patients with various advanced cancers were determined by the inhibition ELISA. The amount of 1‐methyladenosine in urine of 33 healthy donors was 1.91±0.66 nmol/μmol creatinine. In 54% (51/94) of patients, urinary 1‐methyladenosine was elevated above the mean plus 2 standard deviations for the healthy donors (3.23 nmol/μmol creatinine). In patients with leukemia, esophageal cancer, stomach cancer, colon cancer, and bladder cancer, urinary levels of 1‐methyladenosine were significantly elevated. In patients with leukemia, urinary 1‐methyladenosine levels changed almost in parallel with the change in the clinical response during chemotherapy. These results suggest that urinary 1‐methyladenosine might be useful in monitoring the effectiveness of therapy.

Distribution of carbon-11 labeled methamphetamine and the effect of its chronic administration in mice

[11C]Methamphetamine, a psychotropic agent, was synthesized by N-methylation of amphetamine with [11C]CH3 I in hopes that it could be applied in the near future to assist positron emission tomography (PET) in the imaging of its distribution in the human brain. The regional distribution of [11C]methamphetamine was investigated in the mice brain at various intervals after an intravenous (i.v.) injection. Radioactivity was higher in the hypothalamus, cortex, striatum and hippocampus. Furthermore, in chronically administered mice, the uptake of [11C]methamphetamine was higher in the striatum than those in other regions. The regional differences in the distribution of methamphetamine in the mice brain may enable the imaging of its distribution by PET using [11C]methamphetamine.

Monoclonal antibodies against a high spin form of rat cytochrome P-448

Ten monoclonal antibodies reactive with a high spin form of rat cytochrome P-448 (P-448-H) were obtained from hybridoma clones established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells of a BALB/c mouse hyperimmunized with the cytochrome. One monoclonal antibody recognized an epitope characteristic for P-448-H. Five monoclonal antibodies were cross-reactive with a low spin form of rat cytochrome P-448, but not with cytochrome P-450. Reactivity of these monoclonal antibodies with microsomes of rats pretreated with drug metabolizing inducers and Western blots of the microsomal cytochrome P-450 components are also demonstrated.

Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Glycyrrhizin and Its Aglicon, Glycyrrketic Acid

Monoclonal antibodies (MoAbs) specific for glycyrrhetic acid (GA) were prepared and characterized. Obtained MoAbs (AGA-1, AGA-3, and AGA-6) reacted with GA dose-dependently, but not with glycyrrhizin (GL), carbenoxolone, and steroids. Next, an enzyme-linked immunosorbent assay (ELISA) system using AGA-1 was established. The standard curve showed good linearity between 0.01 and 1000 ng/ml of GA, and the detection limit was 5 pg/ml. Recovery, and intra- and interassay variations of this assay system was satisfactory. GL was also measurable quantitatively after acid-hydrolysis of the samples. The developed ELISA system would be useful to determine GL and GA in biological samples or drugs as an alternative method to high performance liquid chromatography (HPLC).

Sensitive detection of two IgG Fc receptors of mouse macrophages by chemiluminescence analysis.

Luminol-enhanced chemiluminescence assay was used to detect the surface expression and the consequent activation of receptors (FcRI and FcRII) of murine macrophages (M phi s). When murine IgG2a was used for the specific detection of FcRI and IgG2b for FcRII, a newly established procedure enabled us to detect the activation of each receptor with as few as 3 X 10(5) M phi s. Briefly, TNP-SRBC coated with monoclonal IgG2a or IgG2b antibodies directed to TNP (sensitized SRBC) were used as reagent, in the presence of 1 X 10(-5) M luminol, and the emission was measured with a liquid scintillation counter. When results obtained by chemiluminescence counting were compared to the results obtained by the rosette formation by adding the same SRBC reagent to peritoneal M phi s obtained after ip injection of Listeria, fortified chemiluminescence counting allowed us to obtain a more definite answer about the activation of each receptor. Under the conditions established, the specific activation of FcRI was obtained by the addition of rIFN alpha A/D to the resident M phi s in vitro and the specific activation of spleen M phi FcRII by iv injection of IAP (Immunosuppressive acidic protein) into mice. These two results supported the independence of the two receptors detected by the assay.

The distribution of [11C]cocaine in normal and cocaine-sensitization mice.

[N-11C-methyl]-cocaine ([11C]cocaine), synthesized by N-methylation of norcocaine with [11C]CH3I, was used to assist in imaging the variety of local distribution by positron emission tomography (PET). The radiochemical yield and the radiochemical purity after purification of [11C]cocaine by high performance liquid chromatography (HPLC) at a sp. act. of 814 GBq/mmol were 47-58% and > 99%, respectively. The time required for synthesis including the purification was 25-30 min from the end of [11C]CH3I trapping. The physical distribution of [11C]cocaine in organ was also investigated in mice at various time after i.v. injection. The main accumulation of radioactivity occurred in the lung, kidney and brain within 1 min after the injection. In the brain, no differences in the organ were observed except the radioactivity level in each section increased for the first 5 min, since then radioactivity decreased dramatically. Furthermore, in the behavioral sensitization model of cocaine, the peak of [11C]cocaine uptake in each brain area was shown to be 5-15 min.

Direct determination of serum glycyrrhetic acid by a monoclonal antibody-based inhibition ELISA using ibuprofen for releasing serum albumin-bound glycyrrhetic acid

We found that ibuprofen (IBU) had a potential for releasing serum albumin-bound glycyrrhetic acid (GA). Based on this observation, IBU was used to pretreat samples for the determination of serum GA levels by an inhibition ELISA. This method, termed IBU method was evaluated by the recovery of GA from human serum albumin (HSA) or normal human serum (NHS) that contained the exogeneously added GA (37-1000 ng/ml). The mean recovery of GA from HSA and NHS samples treated with IBU were 104.7 and 105.2%, respectively, whereas those without IBU pretreatment were 2.8 and 10.7%, respectively. Comparison of IBU method and chloroform extraction method revealed that the GA content of serum samples pretreated by each method were almost the same. These results suggest that IBU method is useful as a serum processing procedure for the determination of serum GA levels by an inhibition ELISA.