Akiyuki Ohkubo

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 Runyons groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyons group III than to that of Runyons group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyons 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.

Identification of three types of PDGF-A chain gene transcripts in rabbit vascular smooth muscle and their regulated expression during development and by angiotensin II

PDGF-like peptides secreted from smooth muscles have been suggested to be responsible for the smooth muscle growth. In order to elucidate the nature of PDGF-like molecules expressed in vascular smooth muscles, we have isolated and characterized cDNA clones for PDGF-A chain from a rabbit embryonic aorta cDNA library. One of the cDNA clones was found to encode a novel PDGF-A chain, named PDGF-A3 in this report. PDGF-A3 arises from a single PDGF-A chain gene by alternative RNA splicing and differs from the sequences of previously reported endothelial- or the glioma-type transcripts by a 110 bp insertion. Expression of PDGF-A3 mRNA was selectively induced by Angiotensin II in the smooth muscle cell in vitro. Total PDGF-A mRNA is most enriched in embryonic aortas, but its expression is down-regulated with vascular development. PDGF-A mRNA is markedly increased in primary-cultured smooth muscle cells during the log-phase growth. Our results suggest that autocrine production of PDGF-A chains from the smooth muscle cell may play a role in early vascular development and in Angiotensin II-induced smooth muscle cell proliferation.

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.

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. J. Clin. Lab. Anal. 11:39–44.

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.

Total urinary protein sensor based on a piezoelectric quartz crystal

A protein sensing system using a piezoelectric sensor with an AT-cut quartz crystal of a basic resonant frequency of 9 MHz has been developed and applied to the determination of total protein in urine. The measurement method is based on the sedimentation of proteins by a turbidimetric procedure. The amount of precipitate formed was determined as the resonant frequency change of the quartz crystal because of the mass change on the surface of the piezoelectric sensor due to the precipitation. A satisfactory correlation was observed between the protein concentration and the frequency change at the range from 50 to 1000 mg/l, and a coefficient of variation (C.V.) of 5.3% (n = 10) was given for 300 mg/l albumin when sulfosalicylic acid was used as the precipitation reagent. When trichloroacetic acid was used as the precipitator, the C.V. was 3.2% (n = 12), and the difference in results for the same concentration of albumin and globulin was much less than that when using sulfosalicylic acid. Treatment with a protease after the measurement was effective for cleaning the electrode surface, allowing the device to be repeatedly used over 300 times.

Changes in Aminolevulinate Synthase and Aminolevulinate Dehydratase Activity in Cirrhotic Liver

Delta-Aminolevulinate synthase and delta-aminolevulinate dehydratase activities were determined in liver biopsy specimens obtained from 12 patients with hepatic cirrhosis. Delta-Aminolevulinate synthase activity was determined by the incorporation of [1,4-14C]succinyl coenzyme A into delta-aminolevulinate. The mean activity of delta-aminolevulinate synthase was significantly higher in cirrhotic liver specimens (mean +/- SE, 193.7 +/- 34.5 picomoles delta-aminolevulinate per milligram protein per 30 minutes) than in controls with minimal histologic changes (32.7 +/- 13.6, p less than 0.01). Furthermore, the mean activity of delta-aminolevulinate synthase was higher in micronodular cirrhosis (281.6 +/- 58.8) than in the other types of cirrhosis (131.0 +/- 23.1, p less than 0.05). Levels of indocyanine green retention at 15 min correlated with the activity of hepatic delta-aminolevulinate synthase (p less than 0.05). The mean activity of delta-aminolevulinate dehydratase, in contrast, was significantly lower in cirrhotic liver specimens (9.4 +/- 1.3 nanomoles porphobilinogen per milligram protein per hour) than in controls (22.0 +/- 2.6, p less than 0.05). These results suggest that the extent of liver injury or the degree of portosystemic shunting, or both, influence the rate of hepatic heme biosynthesis.

Resolution of unresolved peaks containing unknown components by high-performance liquid chromatography with multi-wavelength detection

Abstract A method for the resolution of unresolved peaks obtained by high-performance liquid chromatography with multi-wavelength detection was developed. The method estimates the elution profiles and absorption spectrum of a component eluting at the rising edge or trailing edge of the unresolved peak and estimates the relative intensity of the derived three-dimensional chromatogram of one component by rank annihilation. Artificial unresolved peaks and actual unresolved three-component peaks were resolved by the developed method. The results showed that the method can estimate peak area with errors of less than about 10% when the resolution R s of the components is greater than about 0.4. The accuracy of estimation is considered to be superior to that of the method based on principal component analysis followed by multiple regression analysis, especially if the elution profiles of components are distorted from a Gaussian shape such as with tailing, where the estimation of elution profiles by principal components analysis seems erroneous.

Serial Changes in Cytosolic, Mitochondrial, and Lysosomal Enzymes and Cardiac Myosin Light Chain II in Plasma following Coronary Ligation in Conscious Closed-Chest Dogs

We studied serial changes in various myocardial enzymes and cardiac myosin light chain II (LCII) in plasma following coronary ligation in 14 conscious closed-chest dogs. Cytoplasmic enzymes [creatine phosphokinase (CPK) and supernatant glutamic oxaloacetic transaminase (sGOT)] reached maximum at 12-24 hr and returned to normal at 72-96 hr. The mitochondrial isozyme of GOT (mGOT) began to rise at 6-9 hr, peaked at 12-30 hr (4.8-42.2 IU/liter), and stayed higher at 96 hr than before infarction. Glutamate dehydrogenase (GLDH), another mitochondrial enzyme, began to elevate at 6-16 hr and reached maximum at 24-60 hr (6.2-20.5 U/liter); GLDH also showed higher levels at 96 hr than before infarction. N-Acetyl-beta-glucosaminidase (NAG), a lysosomal enzyme, showed a biphasic pattern in every case. The first peak appeared at 3-12 hr, and the second one at 36-72 hr. Myosin LCII began to rise at 3-9 hr, peaked at 30-120 hr (34-136 ng/ml), and remained elevated for 7 to 10 days. Determination of these myocardial enzymes or LCII in plasma is useful for the diagnosis of acute myocardial infarction.