Site-directed mutational analysis for the ATP binding of DnaA protein. Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.     

Long-term study of patients with pulmonary tuberculosis sequelae after bilateral thoracoplasty-airway obstruction and its causes

Chest roentgenograms and results of pulmonary-function tests in patients with pulmonary tuberculosis sequelae 30 years after bilateral thoracoplasty were studied retrospectively to detect airway obstruction in these patients and to determine its causes. For periods of more than 10 years, vital capacity (VC) changed at a rate of 15.5 +/- 5.0 ml/year, and forced expiratory volume in one second as a percent of VC (FEV1%) changed at a rate of 0.546% +/- 0.380% per year (n = 13). Thirty years after thoracoplasty, the VC was 920 +/- 180 ml (%VC = 28.4% +/- 5.3%), and the FEV1% was 66.2% +/- 13.7% (n = 21). Thus, mild airway obstruction was found in about half of the cases. For each patient, the distance from the hilum to the diaphragm was measured along the mid-clavicular line on the side with fewer ribs resected, and this distance was divided by the patient's height. The results of that computation was found to be significantly and negatively related to FEV1% (r = -0.681, which suggests that longer bronchi in the lower and middle lobes on that side were associated with lower values of FEV1%. These findings are similar to those in patients with pulmonary tuberculous sequelae after total pneumonectomy. Over an average of 26 years, scoliosis, the vertebra showing the most bending, the intrapulmonary lesion, and the position of the diaphragm did not change, but the cardio-thoracic ratio increased.     

Operational Broadcasting Satellite in Japan

The Broadcasting Satellite-2 (BS-2) Program is intended to establish the first Japanese operational domestic satellite broadcasting system using K-band frequencies allocated to Japanese domestic satellite broadcasting service by 1977-WARC-BS. This paper presents the outline of the BS-2 program, including mission objectives, spacecraft, ground system and home receiver.     

Spotted fever group rickettsiosis and vectors in Kanagawa prefecture

Primer pairs for PCR were designed from the gene encoding the 17,000-molecular-weight genus-common antigen of Rickettsia japonica, Rickettsia rickettsii, Rickettsia conorii, Rickettsia typhi and Rickettsia prowazekii. Primers R1, R2 were designed for amplifying the genomic DNA from spotted fever group (SFG) rickettsiae and epidemic typhus rickettsiae. Primers Rj5, Rj10 were designed for amplifying the genomic DNA from only R. japonica. Using the primers R1, R2, about a 540-bp fragment was observed by amplifying the genomic DNA from R. japonica, R. rickettsii, R. conorii, Thai tick typhus TT-118, Rickettsia sibirica, Rickettsia montana, Rickettsia askari, R. typhi, R. prowazekii and Katayama strain isolated from the patient infected with SFG rickettsiae. Using the primers Rj5, Rj10, the 357-bp fragment was observed by amplifying the genomic DNA from R. japonica and Katayama strain. Therefore, the Katayama strain was identified to belong to R. japonica. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp bands were amplified from blood of the patients infected with SFG rickettsiae in Kanagawa prefecture. These findings indicate that the causative agent of SFG rickettsiosis in these two patients was R. japonica. The ticks, Ixodes ovatus and Haemaphysalis flava, were collected by out field research in Kanagawa prefecture. With primers R1, R2 and Rj5, Rj10, 537 bp and 357 bp were amplified from these ticks. This indicates that I. ovatus and H. flava were the vector of R. japonica in Kanagawa prefecture. Also, with the primers R1, R2, about a 540 bp fragment was amplified but with primers Rj5, Rj10, no fragments were amplified from I. ovatus and H. flava. Therefore, these ticks may have SFG rickettsiae other than R. japonica and epidemic typhus rickettsiae.     

3-Dimensional graphic image representation of holographic microscopical displacement pattern by using cubic spline function

For quantitative measurement of microscopical displacement of an object surface due to thermal stress, the holographic pattern measuring system (HPMS) has been developed by the authors, which is combined both the techniques of holographic interferometry and graphic image processing.

In this measuring sytem, a cubic spline function was applied to get the 3-dimensional graphic image patterns of the distribution of the displacement. The displacement is calculated from the interferometry fringe pattern, and the system was applied to deformation analysis of printed circuit board (PCB) due to thermal stress. The PCB deformation was expresed by 3-dimensional graphic image. A smooth curve could be obtained from rather less number of sampled data of interferometry fringe.     

In vivo characterization of T-794, a novel reversible inhibitor of monoamine oxidase-A, as an antidepressant with a wide safety margin

T-794 is a new reversible inhibitor of MAO type A. In order to predict its clinical utility as an antidepressant, we examined its pharmacological profile (i.e., MAO inhibitory activity, antidepressant-like activity and safety) in vivo in rodents. The p.o. administration of T-794 potentiated L-5-hydroxytryptophan-induced symptoms with ED50 = 1.01 mg/kg (mice) or 1.15 mg/kg (rats), and L-dopa-induced behavior with ED50 = 5.90 mg/kg (mice), whereas it did not alter the effect of beta-phenylethylamine even at 100 mg/kg (mice). In the L-5-hydroxytryptophan test in rats, the activity of T-794 (at twice the dose of ED50) disappeared by 8 h; the duration of action was similar to that of moclobemide. These results confirm the previous biochemical results that MAO-A inhibition by T-794 is highly selective and of short duration. T-794 was effective in three animal models of depression: reserpine reversal (mice, rats), behavioral despair test (mice) and learned helplessness (rats). In these tests, it had potency similar to or greater than moclobemide, tranylcypromine or imipramine. The p.o. administration of T-794 (30 mg/kg) did not affect the pressor effect of tyramine in anesthetized rats, whereas moclobemide (30 mg/kg) and tranylcypromine (6 mg/kg) potentiated the effect. Acute toxicity of T-794 proved to be very low (maximal tolerated dose > 2 g/kg p.o.) in contrast to brofaromine (maximal tolerated dose = 150 mg/kg p.o.). Unlike tricyclic antidepressants, T-794 did not prevent the oxotremorine-induced tremor even at 100 mg/kg p.o.; in this it demonstrated a lack of the anticholinergic activity. These results suggest that T-794 is an effective and particularly safe antidepressant and that it may make an important contribution in the treatment of depressive disorders.     

Increased disruption of sperm plasma membrane at sperm immobilization promotes dissociation of perinuclear theca from sperm chromatin after intracytoplasmic sperm injection in pigs

The effects of sperm-immobilization methods on decondensation of sperm chromatin and retention of subacrosomal sperm perinuclear theca (SAR-PT) after intracytoplasmic sperm injection (ICSI) were examined in pigs. Sperm membrane damage caused by different immobilization methods by rubbing with a micropipette without piezo pulses (R), or with a low (L) or high (H) intensity of piezo pulses while rubbing, was assessed by the time required for staining of sperm heads with eosin Y solution. The average time for staining of sperm heads immobilized by the R, L or H treatments was 76, 41 or 26 s, respectively. The fertilization rate following ICSI was increased by sperm immobilization by piezo pulses compared with R, but increased intensity of pulses from L to H did not cause further improvements (29, 48 and 47%, respectively). An immunofluorescence study revealed that H immobilization promoted the dissociation of SAR-PT from sperm chromatin compared with L and R, and it increased the frequency of male pronuclear formation in which chromatin appeared uniformly decondensed. With in vitro fertilization (IVF), SAR-PT disassembled coordinately with sperm chromatin decondensation and it was not detectable around male pronuclei. This was different from most of the oocytes after ICSI in which remnants SAR-PT were detected adjacent to male pronuclei. We concluded that increased damage on the sperm plasma membrane at immobilization improved fertilization rates and decondensation of sperm chromatin after ICSI due to the accelerated dissociation of SAR-PT from the sperm nucleus. Also, the behavior of SAR-PT after ICSI was different from that observed in oocytes after IVF.