Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

[A case report of typical scleroderma accompanied with serum abnormalities characteristic of SLE during the course].

A 40-year-old woman had complained of cyanosis induced by cold exposure from the age of 26. When she was 32 years old, Raynaud's phenomenon occurred. She developed diffuse cutaneous sclerosis affecting the upper limbs, face and trunk, digital pitting scar, flexion contractures of hands, dilatation of lower esophagus and pulmonary fibrosis, and she was diagnosed as scleroderma. Laboratory findings revealed positive anti-topoisomerase I antibody and hypergammaglobulinemia (IgG 2,782, IgA 632, IgM 146 mg/dl). However, serum complement levels were normal and anti-DNA antibodies measured by radioimmunoassay (RIA) were negative. Initial dose of oral prednisolone was 30 mg/day and afterwards 5 mg/day of prednisolone was maintained. At the age of 36, scleroderma and contraction of hands were progressed, and telangiectasias appeared on her chest at the age of 36. Laboratory tests revealed hypocomplementemia (C3 27, C4 9 mg/dl, CH50 16 U/ml) and high titers, more than 100 U/ml, of anti-DNA antibodies measured by RIA. Clinical evidence suggestive of SLE could not be found. Reexamination of previous sera by enzyme immunoassay, in which anti-DNA antibody could not be detected by RIA, clarified the presence of IgG anti-dsDNA antibodies. It was considered that there existed low avidity/affinity of anti-dsDNA antibodies at first, and afterwards high avidity/affinity of anti-dsDNA antibodies appeared. Increasing of oral prednisolone up to 30 mg/day normalized serum complements and decreased titers of anti-DNA antibodies. She had not developed any clinical evidence that suspected SLE throughout the course.

Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains. CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins. To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes. CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined. Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B. stearothermophilus NO2. Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence. Base substitutions were found at the third letter of five codons among the three genes. Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence. The molecular weight of the mature enzyme was estimated to be 75,374. The CGTase gene (cgtM) of B. macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence. The molecular weight of the mature enzyme was estimated to be 74,008. The sequence determined in this work was quite different from that reported previously by other workers. From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B. stearothermophilus NO2 and cgtM from B. macerans IFO3490. We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability. It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.

Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch. The CGTase molecule is composed of four globular domains, A, B, C, and D. In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2. Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F255I, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region. It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme. Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.

[Analysis of chronic excretors of Mycobacterium bacilli in Aichi Prefecture].

Among the patients diagnosed as having pulmonary tuberculosis who were newly admitted to six major tuberculosis centers in Aichi Prefecture during the period from January 1, 1982 to December 31, 1986, 73 patients were chronic excretors of Mycobacterium bacilli (chronics), whose sputum cultures tested positive at both 11 and 12 months after admission. In this study, those 73 patients were analyzed in March 1988. If the patient was still hospitalized at that time, the patient was re-evaluated six months later. The life table method was used for analysis of clinical procedures. For the analysis of risk factors for chronics, 37 patients who were treated after 1971 when RFP was available for treatment were used. The results in this study are as follows: 1. A total of 49.3% of the patients were determined chronics (those who had initial chemotherapy before 1970 when RFP was not available). This result suggests that the use of RFP may contribute to the reduction of chronics. 2. Patient delay in diagnosis, as well as irregular chemotherapy, was one of the factors for the development of chronics. 3. In the chronics, many patients showed severe findings in their chest X-rays, high bacillary counts in the sputum, and low drug-sensitivity bacilli when treated with the major anti-tuberculosis drugs. 4. The rate of chronics from newly admitted patients with positive sputum cultures following the initial chemotherapy treatment was 1.3%. This rate could increase following longer observation periods. 5. Chronics were more prevalent in patients with Type I (extensive cavitary type) findings in the chest X-rays, and positive sputum smears for Mycobacterium bacilli in the clinical findings when compared with all newly registered active tuberculosis patients during the same period in Aichi Prefecture. 6. Concerning the outcome, 55 patients were discharged (17 improved; 13 personally released; 25 deceased) while 18 remain hospitalized. Among those discharged patients, 17 patients were released as a result in improved health while 25 died. These results indicate poor prognosis. Using the life table method, the estimated mortality rate is 49.7%, and the negative sputum rate is 30.5% five years later. 7. Using a multivariate analysis on the prognosis, the risk factor was greatest for poor prognosis for those patients who had Type I findings in their chest X-rays, initial chemotherapy before 1970, complications, and that were male. 8. The patients with improved health, were more sensitive to the drugs applied when compared to the fatal patients. 9. Careful chemotherapy during the first two years may contribute to the reduction of chronics.

[Therapeutic efficacy of imipenem/cilastatin sodium on respiratory tract infections in lung cancer patients].

Imipenem/cilastatin sodium (IPM/CS) was used to treat respiratory tract infections (RTI) in 54 patients with lung cancer. Out of the 54 patients studied, 53 were evaluable for the utility of IPM/CS; 42 had pneumonia, 9 had obstructive pneumonia, 1 had a lung abscess and 1 had acute bronchitis. The efficacy rate was 71.7%. Seventeen causative organisms were isolated from 14 patients. They included Staphylococcus aureus 5 strains, Staphylococcus epidermidis 4 strains, Staphylococcus sp. 2 strains, Enterococcus faecalis 1 strain, Pseudomonas aeruginosa 2 strains, Pseudomonas fluorescens 2 strains, Acinetobacter sp. 1 strain, and the eradication rate was 81.8%. Clinical adverse effects (nausea and vomiting) were observed in 1 patient. Abnormalities in laboratory test results were observed in 3 patients. They disappeared or returned to normal values after completion of therapy or discontinuation of IPM/CS administration. IPM/CS appears to be a useful antibiotic for RTI in patients with lung cancer.

[A case of miliary tuberculosis with prolonged high fever for more than 2 months under antituberculous therapy].

A 28 year-old male was admitted to our hospital with persistent cough and high fever. He was diagnosed to have miliary tuberculosis by the transbronchial lung biopsy specimen and tuberculous choroidal lesions in the ocular fundus. Antituberculosis therapy was immediately started. In spite of the fact that the bacilli were sensitive to the antituberculosis drugs used and he had no other complications, high fever persisted and lasted for more than 2 months. When tuberculosis is suspected, and antituberculosis treatment is tried to observe its clinical response, the presence of similar cases mentioned above should be taken into consideration.

[Antigen presentation in pulmonary tuberculosis].

Antigen presenting capacity (APCC) by monocytes (Mono) and alveolar macrophages (AM), using PPD as the antigen, was determined in 15 patients with pulmonary tuberculosis and 9 healthy controls who all showed positive PPD skin tests. Results were as follows: 1) Mono from both healthy controls and tuberculosis showed APCC to autologous peripheral T lymphocytes, and no significant difference was observed between the two groups. 2) AM from tuberculosis showed APCC to autologous peripheral T lymphocytes, however AM from healthy controls did not. 3) APCC by autologous Mono or AM to lung T-lymphocytes was lower than that to peripheral T-lymphocytes, but the difference was not significant. 4) In tuberculosis, APCC, observed before chemotherapy, was remarkably weakened during the first two months of therapy, and almost recovered to the previous level thereafter. 5) The mechanism which enhances APCC by AM in tuberculosis is uncertain. But neither increased DR antigen expression on AM nor release of IL-1 from AM suggested to be be responsible for the enhanced APCC in tuberculosis.