A new coupling method for the synthesis of oligodeoxyribonucleotides based on protection by protonation of nucleoside bases.

We developed a conceptually new strategy for the coupling method in the phosphoramidite method without protection of nucleoside bases. This strategy, called "proton-block method", significantly reduced side reactions compared with the previous methods.

Studies on thyroid function in rats subjected to repeated oral administration with kojic acid.

To elucidate the effects of kojic acid on thyroid function, the compound was given orally to male rats for 4 weeks at 0, 4, 15, 62.5, 250 and 1,000 mg/kg. In 1,000 mg/kg treatment of kojic acid, the rats showed a slight decrease in motility, inhibition of body weight gain, and a decrease in food consumption. An increase in thyroid weight and a morphological change, i.e., hypertrophy of epithelial cells of the thyroid gland follicles, were observed after 1 week of administration. In addition, the uptake of radioactive iodine from blood into the thyroid gland was enhanced and the TCA-precipitable radioactive iodine in the thyroid gland increased in those rats. However, the rates of the iodination in the thyroid gland did not change during the experiment period. Although serum T4 concentration was low in the rats treated with 1,000 mg/kg kojic acid, it was not observed in any changes in TSH concentration. None of these changes were found in the other groups. These observations suggest that massive administration of kojic acid may decrease blood T4 concentration and that thyroid function may be enhanced compensatorily. On the other hand, the absorption of kojic acid was rapid as manifested by the Tmax of blood concentrations of radioactivity, which was as short as 1.0 +/- 0.0 hr, and the t1/2 was 4.8 +/- 0.3 hr. Blood concentrations of radioactivity disappeared nearly completely at 24 hr after administration. This result indicates that the toxic effect observed on the thyroid gland treated with only the largest dosage of kojic acid may depend on a fast decrease following a transient increase of concentration of the compound in the blood.

[Feasibility of a simple measurement for white blood cell by immunologically measured granulocyte elastase in whole blood].

Using whole blood sample, we examined the correlation between concentration of granulocyte elastase (GEL) in granulocyte obtained by immunoassay and white blood cell counts. The correlation between concentration of GEL and granulocyte cell counts was also examined. The correlation coefficient between concentration of GEL and white blood cell counts was R = 0.87, and that between concentration of GEL and granulocyte cell counts was R = 0.91. The correlation coefficients of outpatients and patients with diseases accompanied by inflammation except for tumors were better than those of inpatients. The GEL concentration in granulocyte using whole blood sample well responded to the white blood cell counts against the tolerance for a short time, such as after operations. Especially on screening tests of diseases accompanied by inflammation and primary care, analysis of GEL in whole blood is not only useful for observation of inflammation and its progress but also suggests possibility of converting GEL to white blood cell counts or granulocyte. Furthermore, because it can be measured by easy-operative latex agglutination turbidimetric method, an easy measurement system can be built. Extended usage of the system as a rapid diagnostic tool on emergency tests in general clinics and hospitals are expected.

Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Acute exacerbation of autoimmune liver disease associated with hantaviral infection.

Hantavirus is known to cause haemorrhagic fever with renal syndrome (HFRS). Although liver dysfunction has been reported in HFRS, hepatic manifestations of hantaviral infection have not been well described. We describe a case of autoimmune cholangitis in which an exacerbation of hepatitis was associated with hantaviral infection. Seroconversion of both IgG- and IgM-class antibodies to hantavirus was noted coincident with acute exacerbation of hepatitis, which was resolved promptly by treatment with corticosteroid. No extrahepatic manifestations were noted. This case suggests that hantavirus may trigger acute exacerbation of autoimmune liver disease without extrahepatic manifestations and that it may cause community-acquired hepatitis.

High prevalence of hantavirus infection in a group of Chinese patients with acute hepatitis of unknown aetiology.

In southwestern China, small but substantial numbers of patients with acute hepatitis were found without known hepatropic viral infections (hepatitis A, B, C, D or E, cytomegalovirus, Epstein-Barr virus) and were receiving no hepatotoxic drugs. Prevalence of antibodies, both neutralizing and specific immunoglobulin (Ig)M and IgG, to Hantaan virus were evaluated in a cohort of 136 such patients: 83 were of unknown aetiology, 53 had known viral hepatitis and 59 healthy subjects acted as controls. The results showed that the incidence of neutralizing antibody to Hantaan virus in acute hepatitis patients with non-hepatitis A-E virus infections (13 of 83) was significantly higher than in those with A-E infections (0 of 53, P<0.01). Furthermore, the incidence of specific IgM antibody to Hantaan virus in acute hepatitis patients with non-hepatitis A-E virus infections (6 of 83) was significantly higher than in those with A-E infections (0 of 53, P<0.05) and in healthy subjects (0 of 59, P < 0.05). These findings suggest that Hantaan virus may be an important agent, contributing, at least in southwestern China, to a significant number of the cases of acute hepatitis of unknown aetiology. This hantavirus infection resulted in an acute hepatitis, differing from the typical diseases: haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS).

Serum protein standardization project in Japan: evaluation of an IFCC reference material (RPPHS/CRM470) and establishment of reference intervals.

Reference preparation for proteins in human serum (RPPHS), also called Certified Reference Material 470 (CRM 470), was prepared by the international Federation of Clinical Chemistry (IFCC) and is intended to serve as a new international plasma protein reference material. It is now being introduced into Japan. RPPHS possesses many excellent properties, including safety, stability, and accuracy in value assignment. Moreover, the physicochemical properties of its proteins are identical to those of fresh serum, giving it immunochemical behavior that is commutable with that of existing reference materials and calibrators in given immunoassays. Reference intervals of 13 serum proteins were determined for the first time using nephelometry and a new working calibrator assigned from RPPHS, which seems certain to play a critical role in the global standardization of specific protein immunoassays.

[Establishment of reference intervals and physiological parameters for 13 serum proteins in healthy Japanese adults].

Using a nephelometry with a new calibrator in which values were assigned from EU/CRM470, a new international reference preparation, we have established for the first time the reference intervals of 13 different serum proteins in Japanese adults. Based on the NCCLS guideline C28-P, a new statistical and concrete procedure was revised and used for this study. It is clearly shown here that standardisation of serum proteins can be possible under the qualified assay system, making it possible to share the reference intervals among laboratories under well-controlled quality assurance. New aspects of serum protein variations were discovered in relation to physiologic changes and life styles.

Differentiation of beating cardiac muscle cells from a derivative of P19 embryonal carcinoma cells.

A clonal derivative named CL6 has been isolated from pluripotent P19 embryonal carcinoma (EC) cells, after long term culture under conditions for mesodermal differentiation. The CL6 subline has a morphology similar to P19 cells and grows exponentially in standard medium, but unlike P19 cells, it efficiently differentiates into beating cardiac muscle in adherent culture with 1% dimethyl sulfoxide (DMSO). During differentiation, CL6 cells displayed the following general cardiac muscle properties: (1) alpha- and beta-cardiac myosin heavy chain (MHC) transcripts were first detected on day 10 after treatment with DMSO. (2) A sarcomere MHC protein also appeared on day 10, simultaneously with the initiation of contraction. (3) Immunofluorescence analysis showed that vigorously contracting cells have a striated structure. (4) Skeletal muscle specific transcripts, such as myogenin and MyoD, were not detected throughout differentiation. On the other hand, in suspension culture with 1 microM retinoic acid (RA), the condition for neural differentiation of P19 cells, the CL6 cells developed into neurons with poor outgrowth. Taken together, the CL6 subline seems not to be committed to a mesodermal lineage but to represent a developmental stage closer to differentiated cardiac muscle than the P19 cell line. Since CL6 cells directly differentiate into cardiac muscle in adherent culture, it is the most useful cell line for studying the differentiation of cardiac muscle in vitro.

Acidic catecholamine metabolites and 5-hydroxyindoleacetic acid in urine: the influence of diet.

Concentrations of vanillylmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), vanillic acid (VA) and 5-hydroxyindoleacetic acid (5-HIAA) in urine from healthy subjects were determined by a high-performance liquid chromatography system with a mixed-mode (C18/anion-exchange) column and an 8-channel electrochemical detector, in order to study the influence of diet, diurnal variation and age. The urinary excretion of 5-HIAA increased significantly after eating banana, pineapple, tomato, kiwi fruit and walnut. An increase in the urinary excretion of DOPAC and HVA after eating banana and that of VA after taking vanilla was also noted. The urinary excretion of VMA was not significantly influenced by any of the foods examined. The urinary excretion of 5-HIAA in the first-morning urine increased beyond the upper limit of the reference value when banana was taken at 2000 h the previous day. The excretion of all metabolites in the second-morning urine in the fasting state was within respective reference ranges. Diurnal variation of the excretion of VMA, DOPAC, HVA and 5-HIAA in urine was relatively small, but that of VA was large. The concentrations (mmol/mol creatinine) of VMA, DOPAC, HVA, 5-HIAA and VA in the first-morning urine from healthy subjects increased from 7 days after birth to 1 year and then decreased to adult levels at 13 years of age.

[Strategy for getting many experts in clinical pathology].

Laboratory tests are indispensable to rational medical care and will play more and more important roles in clinical and preventive medicine. However, progress in laboratory medicine is so rapid that many clinicians feel difficulty in using laboratory tests properly. To keep the quality of medical care high in terms of medical science, many well-trained clinical pathologists and medical technologists are needed to help clinicians use laboratory tests properly. To obtain enough experts in the area of clinical pathology, we should pave the way to clinical pathology for young ambitious people and make efforts to help both young clinical pathologists and clinical technologists feel themselves playing an important role in medical care.

Simultaneous determination of catecholamines, their basic metabolites and serotonin in urine by high-performance liquid chromatography using a mixed-mode column and an eight-channel electrochemical detector.

A high-performance liquid chromatographic method for simultaneous determination of free catecholamines, their basic metabolites and serotonin in human urine was developed. The compounds were separated on a precolumn of cation-exchange resin and a mixed-mode (C18/cation-exchange) column and determined by an eight-channel electrochemical detector. By this method, free norepinephrine (NE), epinephrine (E), dopamine (DA), normetanephrine (NMN), metanephrine (MN), 3-methoxy-tyramine (3-MT) and serotonin (5-HT) were determined in 40 min with inter-assay precision of 1.9, 3.2, 1.2, 1.6, 1.6, 5.5 and 3.2%, respectively. Detection limits were between 0.05 (E) and 0.3 (3-MT) pmol. The mean values of analytical recovery for NE, E, DA, NMN, MN, 3-MT, 5-HT were 99, 104, 104, 99, 102, 109 and 99%, respectively.

[Reducing unnecessary laboratory use through education and regulation].

Elimination of unnecessary laboratory use is becoming more and more important in the control of the rapid growth of medical costs in Japan. Among several measures to reduce unnecessary laboratory use, advising doctors about rational use of clinical laboratory is effective but not sufficient. At Tokyo University Hospital we have established a system to reduce unnecessary laboratory use. In this system warning about excessive use of laboratory tests appears on the display when a doctor orders more laboratory tests by the console than the maximum number of tests covered by health insurance. With this warning system we can effectively minimize unnecessary use of laboratory tests.

[Effects of kanamycin on platelet satellitism and leukocyte adhesion phenomena].

To examine the effects of kanamycin on platelet satellitism and leukocyte adhesion phenomena, we added kanamycin to the blood sample which showed the platelet satellitism and the leukocyte adhesion phenomena. We observed the rapid dispersion of platelets from the surface of leukocyte in platelet satellitism and also the rapid dispersion of leukocyte in leukocyte adhesion by addition of kanamycin (20 mg) in 1 ml of EDTA-anticoagulated blood from a patient with both phenomena. Neither adhesion of platelet nor adhesion of leukocyte was found on microscopical examinations after dispersion. Complete blood cell counts and their histogram patterns after dispersion were almost the same as those just after venipuncture. The dispersion was found just after the addition of kanamycin and no re-adhesion was observed. These findings suggest that kanamycin leads to dispersions of leukocyte-leukocyte, platelet-platelet and leukocyte-platelet adhesion phenomena.

Clinical usefulness of CYFRA assay in diagnosing lung cancer: measurement of serum cytokeratin fragment.

We evaluated the diagnostic usefulness of measurement of the soluble cytokeratin 19 fragment, a new tumor marker, in 391 patients with lung cancer and in 424 patients with benign lung diseases. Serum concentrations of cytokeratin 19 fragment were measured by a sandwich ELISA (CYFRA). The cut-off value was defined as 3.5 ng/ml, which is associated with a specificity of 85% for benign lung diseases. CYFRA had a high sensitivity (57.5%) in all subjects with lung carcinoma, and had a higher sensitivity for squamous cell carcinoma (73.1%, n = 141) than squamous cell carcinoma-related antigen (61.0%). CYFRA was associated with a relatively high sensitivity (42.1%) in early-stage squamous cell carcinoma (stage I, based on the classification of the Japan Lung Cancer Society), but the CYFRA titer was higher in advanced squamous cell carcinoma than in early-stage squamous cell carcinoma. Our findings suggest that CYFRA is potentially useful for diagnosis and monitoring of lung carcinoma, especially for squamous cell carcinoma.

High-performance liquid chromatographic determination of catecholamine metabolites and 5-hydroxyindoleacetic acid in human urine using a mixed-mode column and an eight-channel electrode electrochemical detector.

An HPLC system for the simultaneous determination of acidic catecholamine metabolites, related compounds and 5-hydroxyindoleacetic acid (5-HIAA) in human urine was developed. A mixed-mode (C18/anion-exchange) column with isocratic elution using citrate buffer and an eight-channel electrochemical detector were used. Vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenyllactic acid (vanillactic acid, VLA), homovanillic acid (HVA), vanillic acid (VA) and 5-HIAA in urine were determined simultaneously. Detection limits and inter (n = 5) and intra-assay (n = 5) coefficients of variation were satisfactory. The mean of analytical recoveries (n = 3, +/- C.V. (%)) were between 97 +/- 3.2 (VMA) and 105 +/- 4.8 (VA). Correlations between the analytical results for VMA, HVA and 5-HIAA obtained by an established method and the present method were satisfactory. The mean +/- 2 S.D. of the excretion rates of VMA, DOPAC, VLA, HVA, 5-HIAA and VA in urine from healthy adult volunteers were 0.61-4.36, 0.13-1.02, 0-0.35, 0.67-6.55, 0.50-5.14 and 0-0.55 mg/g creatinine, respectively.

Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species.

We recently reported a genus-specific PCR for the mycobacterial dnaJ gene. In the present study, we have determined the nucleotide sequences of the dnaJ gene from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. marinum, M. kansasii, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonae, M. hemophilum, and M. paratuberculosis). On the basis of the amplified dnaJ gene nucleotide sequences, we constructed a phylogenetic tree of the mycobacterial species by using the neighbor-joining method and unweighted pairwise grouping method of arithmetic average. We found that the phylogenetic relationship inferred within the slowly growing species was in good agreement with the traditional classification, with three major branches corresponding to Runyon's groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyon's group III than to that of Runyon's group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyon's group IV, indicating that our phylogenetic analysis based on the dnaJ gene reflects the phenotypic characteristics such as pigmentation but not the growth rate. Finally, we revealed the species-specific restriction sites within the amplified dnaJ gene to differentiate most of the mycobacterial DNA by a combination of PCR with restriction fragment length polymorphism analysis.

[Bilirubin metabolism in liver cirrhosis].

The serum bilirubin concentration is a specific marker of liver disease but the sensitivity is low for detecting liver damage and remains within normal limits in many patients with compensated liver cirrhosis. In liver cirrhosis, portal blood flow is distorted accompanied by a decrease in hepatic clearance of bilirubin. In addition, portosystemic shunting as well as splenomegaly results in an increase in hemolysis and production of bilirubin. These together result in an increase in the concentration of unconjugated bilirubin in serum. In advanced cirrhosis, glucuronyl conjugation of bilirubin and biliary excretion of conjugated bilirubin are markedly impaired and jaundice appears. The concentration of bilirubin in serum thus become a good prognostic marker for patients with incompensated liver cirrhosis.

[Detection of anticardiolipin antibody using the EIA kit prepared to eliminate interference of serum cofactor].

We studied sera from patients with various disorders including collagen diseases, Buerger's disease, deep vein thrombosis, repeated abortions and idiopathic thrombocytopenic purpura in order to measure anticardiolipin antibody (aCL) titers. In our assay system, we can detect aCL against bovine cardiolipin and the complex of cardiolipin and beta 2-glycoprotein I (beta 2-GPI). This was done with simultaneous assays using blank wells and bovine cardiolipin coated wells in the EIA method in which both wells were also coated with bovine serum albumin possibly containing beta 2-GPI, then aCL titers was given by subtraction of O.D. values of blank wells from those of cardiolipin coated wells. When the aCL was quantified by anti human immunoglobulin antibodies, collagen diseases showed positive aCL in 15 (11.5%) out of 130 sera with positive anti ENA antibodies, 3 (11.1%) out of 27 sera with positive anti DNA antibodies and 3 (5.2%) out of 58 sera with other positive ANA sera. The other hand, we found positive aCL in 2 (4.9%) out of 41 sera from patients with Buerger's disease, 4 (36.4%) out of 11 sera from patients with deep vein thrombosis, 1 (7.7%) out of 13 sera from patients with repeated abortions and 6 (14.6%) out of 41 sera from patients with idiopathic thrombocytopenic purpura. Twenty one (61.8%) out of these aCL positive sera had also positive IgG-aCL for the assay using anti human IgG antibody. Compared to previous reports, we thought, low incidence of aCL in our study was due to exclusion of anti beta 2-GPI antibody in our assay system.

Establishment of an antitoxoplasma state by stable expression of mouse indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading enzyme, is inducible by various interferons (IFNs). IDO-mediated tryptophan degradation, but not the formation of IDO-catalyzed tryptophan metabolites, has been suggested as a mechanism for the antiparasitic action of IFN-gamma. To determine whether the IFN-gamma-induced IDO alone is sufficient for establishing the antiparasitic state, we constructed a mouse IDO expression plasmid containing a heavy metal-responsive metallothionein promoter and obtained a stable transformant (C6) by transfection of this plasmid into mouse rectal cancer (CMT-93) cells. In the presence of 100 microM ZnSO4, C6 cells yielded a high level of IDO; and after a 2-day culture period, the enzyme induction resulted in complete depletion of tryptophan from the culture medium. Under these conditions, the growth of Toxoplasma gondii in C6 cells infected with the organisms on day 3 after enzyme induction was completely blocked. In the absence of ZnSO4, however, IDO induction was negligible in C6 cells, and T. gondii continued to grow. Furthermore, in a transformant (CC10) carrying an antisense mouse IDO plasmid or in parental CMT-93 cells, IDO was not induced at all even in the presence of 100 microM ZnSO4, and T. gondii continued to grow in these cells as well. These results taken together indicate that complete depletion of tryptophan from the culture by IDO alone is sufficient to establish the antitoxoplasma state in mouse cells.