Various plasmids harboring the truncated DNA polymerase gene (
polA) from
Thermus thermophilus HB8 (
Tth polymerase) were constructed. The most thermostable
TthΔ
NF2 polymerase the gene product of
...polAΔ
NF2, which lacked a 751-bp region (region flanked by initiation codon and
FspI site in the
polA gene) was selected, and purified from the recombinant
Escherichia coli. SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the
TthΔ
NF2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the
TthΔ
NF2 polymerase was 63% of that of the
Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the
TthΔ
NF2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the
TthΔ
NF2 and
Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.
Properties of F. VIII/vWF in highly-purified factor VIII concentrates were examined using monoclonal antibodies. The F. VIII: C levels obtained with the chromogenic assay agreed with those obtained ...with the one-stage clotting method. The ratio between F. VIII: Ag and F. VII: C in Hemofil M and Monoclate was 1.09 and 1.34, respectively. The F. VIII: Ag levels assayed by monoclonal ELISAs were the same as those assayed by polyclonal ELISA, except that those assayed by C 5-ELISA tended to be higher. The ratio between F. VIII: Ag and vWF: Ag in Hemofil M and Monoclate was 105 and 45, respectively. Both concentrates lacked the large multimers of vWF and showed the intensification of the satellite bands. SDS-PAGE patterns showed almost no contamination. Immunoblot analysis revealed that F. VIII in both concentrates could react with 6 kinds of monoclonal antibodies to F. VIII. These results suggest that the fundamental structure of F. VIII molecule for coagulant activity in both concentrates are preserved.
A MEMR syndrome occurred in a 9-year-old boy with cerebral atrophy and was established by computed tomography scans of the cranium. The patient had mental retardation, delayed speech development and ...microcephaly, and high levels of urinary dermatan sulfate and heparin sulfate, which are characteristics of this inborn error of mucopolysaccharide metabolism.
BACKGROUNDCoronary angiography (CAG) has been the mainstay of diagnostic image analysis for coronary artery fistula (CAF). However, it is difficult to fully delineate this complex vessel structure ...including coronary trees, particularly in cases with large CAF, by this method. CASE REPORTSIn the present 3 cases with large CAF, contrast-enhanced multidetector computed tomography (MDCT) was performed to examine the whole coronary vessel structure including CAF. Selective CAG was also undertaken. In all 3 cases, based on the echocardiographic findings and the characteristic heart murmur, presence of CAF was suspected. However, transthoracic echocardiography as well as CAG alone could not define the whole abnormal vessel structure precisely. Moreover, CAG could not obtain clear images of the coronary artery with large CAF, because of contrast-steal. In contrast, MDCT could not only define CAF in detail but also depict coronary artery adjacent to CAF. On the basis of the MDCT findings, in cases 1 and 3, surgical exclusions were undertaken without and with coronary artery bypass grafting, respectively. CONCLUSIONSContrast-enhanced MDCT might be useful for the diagnosis of large CAF and for the estimation of the coronary artery adjacent to CAF, which is absolutely indispensable for surgical treatment.
Properties of factor VIII (FVIII) in recombinant FVIII concentrates (rFVIII, BAY w 6240) were investigated using 6 kinds of monoclonal antibodies to FVIII. The FVIII: C levels obtained by the ...one-stage method using severe hemophilia A plasma as a substrate were identical with those indicated on the label (100u/ml). The FVIII: C levels obtained by the chromogenic method were relatively higher than those by the one-stage method. The FVIII: Ag levels assayed by polyclonal ELISA agreed with those by NMC-VIII/1 (recognizes the 80KDa fragment of L-chain) or NMC-VIII/5 (the 70KDa of L-chain) monoclonal ELISA, whereas the FVIII: Ag levels by C 5 (the 54KDa of H-chain) monoclonal ELISA were 2.5-3.0 times higher than those by other monoclonal ELISAs. The ratio between FVIII: Ag and FVIII: C was 1.4. It was considered that FVIII in the recombinant concentrates lost its activity less than FVIII in the heat-treated concentrates commercially available. No von Willebrand factor was detectable by the sandwich ELISA system. SDS-PAGE pattern showed almost no contamination except albumin as a stabilizer. Immunoblot analysis revealed that FVIII in recombinant concentrates could react not only with monoclonal antibodies to L-chain and H-chain, but also with monoclonal antibodies to middle portion of FVIII molecule. These results suggest that the fundamental structure of FVIII molecule for coagulant activity is preserved in the rFVIII concentrates, and that it is expected to be useful for the treatment of hemophiliacs.
Activated prothrombin complex concentrate (APCC), a by-passing hemostatic preparation for hemophilia A patients with a high titer of factor VIII inhibitors, often has an unpredictable hemostatic ...effect and occasionally results in severe thromboembolic complications. A clinical trial was conducted on 3 hemophilic patients with a high titer of factor VIII inhibitors to evaluate the safety and efficacy of a recombinant factor VIIa (rFVIIa; NN-007), an alternative by-passing preparaton. At the same time thrombelastgram (TEG) was evaluated to be better than activated-partial thromboplastin time (APTT) as a monitor of hemostatic effects when using rFVIIa. TEG was more suitable than APTT, because r, r+k and ma values of TEG were normalized at least for 4 hours after the infusion, whereas APTT was variably shortened and was not always maintained at a normal level for 4 hours. Sixty five μg/kg to 80μg/kg of rFVIIa was administered on demand once to 12 times per one bleeding episode. Eighty bleeding episodes were assessed during the 36-week treatment period. The efficacy rate of the hemostatic effect was confirmed to be 97.5%. No adverse reactions including laboratory or clinical DIC were observed in a total of 123 infusions. No anamnestic response of the factor VIII inhibitor developed. Vital signs and laboratory findings showed no significant changes attributable to rFVIIa. These findings suggest that rFVIIa is safe and efficacious as a by-passing hemostatic preparation for hemophilia A patients with a high titer of factor VIII inhibitors.
Seismic waves from explosions at nine sites were observed at a number of temporary stations in the southwestern Kanto plain. Travel time analyses were carried out along the several surveying lines, ...so that the contradiction at cross points of the lines becomes as small as possible. Underground structures along the surveying lines were studied to reveal the three dimensional features of the region. The results are summarized as follows: 1) The structure consists of four layers which have P-wave velocities of 1.8, 2.8, 4.8 and 5.5km/s, respectively. Around the Yumenoshima explosion site, however, the thickness of the 4.8km/s layer seems to be very thin. 2) The depth to the 4.8km/s layer becomes largest around Yokohama as more than 4km. The thickness of this layer is more than 3km in the central part of the area considered, such as Okazu and Hiratsuka. However, it becomes shallower near the Kurokawa and Higashi-ohgishima explosion sites. 3) A step-like structure was found on the top of the 5.5km/s layer at points along the several surveying lines in the southeastern extension of the Tachikawa fault toward the Tokyo Bay area. On the southwestern side of the step-like structure, the depth to the 5.5km/s layer is more than several kilometers and this layer exists near the ground surface of the Kanto Mountains.
Fe-EDTA concentrations of culture solution in hydropnics were changed for improving the quality of herbs, peppermint, sage, perilla (Perilla frutescens Britton), basil, hyssop and Japanese honewort ...(Cryptotaenia japonica Hassk). Culture solution contained Ca (NO3) 2·4H2O : 45g, KNO3 : 36g, MgSO4·7H2O : 22.5g, NH4HPO4 : 6.75g in 45l water. High concentration of Fe-EDTA (0.296g/e) inhibited the growth of these herbs. The high Fe-EDTA (0.296g/l) concentration with peppermint resulted in the higher contents of chlorophyll, o-diphenol, total phenols and free amino acid than the control (0.148g/l).
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