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Amino acid residue 56 of the phosphatidylserine synthase of Bacillus subtilis was changed from Ser to Pro by using modified primers in PCR amplification of its structural gene, pssBS. When an Escherichia coli mutant lacking its own phosphatidylserine synthase harbored a plasmid carrying this allele, the Mn(2+)-requiring Bacillus-type synthase activity, as assayed in vitro, was at least six-fold lower than that with the wild-type pssBS gene and the cellular phosphatidylethanolamine content was similarly lowered, indicating that the altered region of the enzyme is critically important for its activity. In contrast to the E. coli counterpart, amplification of the wild-type Bacillus enzyme increased both the relative and absolute contents of phosphatidylethanolamine and impaired cell growth. However, amplification of the mutant enzyme of the same level was much less toxic, implying that E. coli cells are more sensitive to the unbalanced accumulation of phosphatidylethanolamine than that of the hydrophobic enzyme molecules. Possible roles of the conserved region of the enzyme in its activity and the wild-type phospholipid composition in the proper membrane function are discussed.

1 Dept. of Civil Eng., Tokyo Institute of Technology (1-12-1 Ookayama, Meguro-ku, Tokyo 152-8552, Japan) E-mail:zhang.w.ac@m.titech.ac.jp Member of JSCE, Dept. of Civil Eng., Tohoku University (6-6-06 Aramaki Aza-Aoba, Aoba-ku, Sendai City, Miyagi 980-8579, Japan) E-mail: matsuzaki@civil.tohoku.ac.jp Member of JSCE, Professor, Dept. of Civil Eng., Tokyo Institute of Technology (1-12-1 Ookayama, Meguro-ku, Tokyo 152-8552, Japan) E-mail: kawashima.k.ae@m.titech.ac.jp

The Escherichia coli pgsA3 mutation, which causes acidic-phospholipid deficiency, was found to repress the flagellar master operon, as assessed by the beta-galactosidase activities of flhD-lacZ fusions. This explained the impairment of flagellar formation and motility by the mutation. A series of deletion analysis indicated that a 40-bp region, at the 5' end of the flhD locus examined, was responsible for the repression of a downstream transcription initiation that was catabolite-repression sensitive. This novel regulatory region was 200 bp upstream of the first possible translation initiation site.

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.

The nucleotide sequence of the Escherichia coli pgsA gene, encoding phosphatidylglycerophosphate synthase, is revised to code for an enzyme of 182 amino acid residues, instead of the 216 of a previous work (A. S. Gopalakrishnan, Y.-C. Chen, M. Temkin, and W. Dowhan, J. Biol. Chem. 261:1329-1338, 1986). The revised structure now explains the properties of the enzyme. Three pgsA mutants of different phenotypes were also analyzed: pgsA3, pgsA36, and pgsA10 have single-base replacements in codons 60 (Thr-->Pro), 1 (ATG-->ATA), and 92 (Thr-->Ile), respectively.

Objective The efficacy of serum pepsinogen (PG) test is widely accepted as a screening test to select persons for endoscopy in the diagnosis of gastric cancer. In this study, we would like to examine whether serum PG levels give us information on endoscopic findings of gastric mucosa. Materials and methods The serum level of PG++ and PG+− and the PG+/PG− ratio were compared with endoscopic 13C-urea breath. H.pylori status was defined as an increase in the intragastric 13CO2/12CO2 ratio of 10% over baseline. Intestinal metaplasia was made visible as the purple-stained area using a 0.05% crystal violet spraying method. PG level of less than 70μg/L and I PG+/PG− ratio of less than 3 was adopted for a (+) result, and PG level of less than 30μg/L and a PG+/PG− ratio of less than 2 for a (++) result. Results Prevalence of endoscopic features and H. pylori infection in different groups classified by serum PG tests. Conclusions Lintestinal metaplasia was identified in more than 80% of PG positive patients. The prevalence of linear reddness and raised erosion in the antrum were higher in PG (-) group than in PG(+) and (++) groups. H. pylori-positive rate was the highest in PG (+) group.

Objective It would be desirable to diagnose H. pylori-related gastritis simply by endoscopic observation. However, it remains controversial whether certain endoscopic features can be used to diagnose H. pylori infection. Material & Methods A new modified endoscopic 13C-urea breath test (Dig Endosc 12:29-32,2000) was performed in 628 patients. Briefly, after endoscopic observation, 20ml of water containing 100mg of 13 (urea is sprayed onto the gastric mucosa. Thereafter, a sample of 150mlof intragastric gas is collected through a biopsy channel. A positive test was defined as an increase in the ratio of 10 per mil over baseline. Endoscopic features were evaluated by the presence of following findings; linear reddness, raised erosion, haemorrhagic erosion, haemorrhagic erosion, and spotty erythema. Results The sensitivity, specificity, positive predicts value (PPV), and negative predict value (NPV) of each feature is shown in the following table: sensitivity specificity PPV NPV spotty erythema (body) 35.9% 89.6% 83.6% 48.7% haemorrhagic 7.6% 86.5% 45.2% 38.8% linear reddness (antrum) 2.7% 69.3% 11.5% 32.6% (body) 2.4% 95.2% 42.9% 39.8% raised erosion (antrum) 14.1% 73.3% 43.7% 36.7% (body) 1.1% 99.6% 80.0% 40.6% Conclusion Spotty erythema in the gastric body was thought to reflect H. pylori infection. In contrast, it was suggested that linear reddness in the antrum indicated the absence of H. pylori.

An improved scanning total reflection (STR) method is discussed to accurately measure the refractive index distribution and dispersion of the gradient index optical element. We also describe the measuring apparatus and experimental results. The accuracy of 1×10-4 (rms) was attained in terms of the absolute measurement of refractive indices, and good reproducibility of the index and Abbe number were also confirmed, δn=1×10-4 (rms) and δνd=0.5 (rms), respectively. A very good agreement is also observed between the result obtained by our method and that by an interference method.

Raman spectra were measured for cac-strain mouse (Nakano mouse) lenses in the various stages of cataract formation. The spectra were compared with those of normal mouse lenses of the corresponding ages. A significant difference was observed in the intensity of the Raman band due to an OH stretching mode of lens water (3390 cirri) between the spectra of cataractous lenses and those of normal lenses. The difference was already obvious in the very incipient stage of hereditary cataract and became more pronounced with cataract development. These observations clearly show that changes in lens water occur during cataractogenesis. We propose in this communication that the intensity change of the Raman band at 3390 cm−1 may be very useful for the diagnostic marker of cataractous signatures.

Two acid phosphodiesterases (phosphoric diester hydrolase, E.C. 3.1.4) were purified to apparent homogeneity from cultured cells of Nicotiana tabacum var. Bright Yellow. These enzymes appeared to differ in some physicochemical properties but exhibited similar broad substrate specificities towards cyclic and p-nitrophenyl nucleotides, and bis-p-nitrophenyl phosphate. Of these compounds, the most active substrates were bis-p-nitrophenylphosphate and 2',3'-cyclic nucleotides. Both p-nitrophenyl 3'- and 5'-thymidylates were hydrolyzed at comparable rates, whereas the hydrolysis of 3',5'-cyclic AMP occurred preferentially at the 5'-phosphodiester bond. A unique catalytic property of these acid phosphodiesterases was the ability to liberate inorganic phosphate from ATP, ADP and p-nitrophenyl phosphate. Ribonucleic acid, its breakdown products, and inorganic phosphate were found to be effective as inhibitors.

When cultured in the presence of 600 mM D-mannitol, Escherichia coli K-12 cells synthesized two novel phospholipids. The identities of these compounds are postulated to be phosphatidylmannitol and diphosphatidylmannitol, the sugar alcohol analogs of phosphatidylglycerol and cardiolipin, respectively. The nonacylated glycerol moieties of the normal acidic phospholipids were substituted by D-mannitol. The formation of the analogs was significantly enhanced when strains harboring the pss-1 allele, a temperature-sensitive mutation in phosphatidylserine synthase (Ohta and Shibuya, J. Bacteriol. 132:434-443, 1977), were grown at 42 degrees C, and the accumulation of the analogs was maximum in late stationary phase; more than 90% of the total cellular lipids were these novel phospholipids. Strains with a defective cardiolipin synthase (Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) failed to form the analog lipids, whereas cells with increased cardiolipin synthase activity due to the presence of a pBR322-derived recombinant plasmid containing the structural gene for cardiolipin synthase produced more mannitol lipids than wild-type strains. These observations and the structures of the analog lipids indicated that cardiolipin synthase participates in the formation of these novel phospholipids. We suggest that reversible alcoholysis and condensation, in addition to low substrate specificity of the enzyme, are the mechanisms involved in this process. Addition to the medium of other straight-chain alditols, D-arabitol, ribitol, xylitol, erythritol, and L-threitol also yielded pairs of novel phospholipids, whereas sorbitol or galactitol produced only one analog in small quantities. These acidic phospholipid analogs have not been reported in any living system. They should be useful in the study of structure-function relationships of phospholipids and in manipulating the structures of various membrane systems.

Existing replica methods designed for adherent somatic cells are not applicable to non-adherent cells of tumor origin. We have developed a new, efficient replica method for the negative selection of mutants of non-adherent mouse FM3A cells. Mutagenized cells are grown clonally on soft-agar plates then allowed to proliferate upwards through layered polyester disks, thus producing replicas with the same colony patterns. Isolation of mutants with increased requirements for myo-inositol by this method is described.

The localization of acid phosphatase activity has been investigated ultrastructurally in regenerating human fetal skeletal muscle cells. The heavy reaction product was observed in the cisternae of Golgi apparatus and lysosomal vesicles, whereas the slight reaction product was also occasionally found in the nuclear envelope, endoplasmic reticulum and sarcoplasmic reticulum of the regenerating myocytes. The enzymatic basis for these deposits was confirmed by their absence in the incubation of the specimen in substrate-free medium and the enzyme inactivation of the specimen by exposure to NaF or heat.

Bacillus natto IAM 1212 produces α-amylase by about 5 times as much as Bacillus subtilis Marburg strain, α-amylase of which is more thermostable and moves more rapidly to anode at pH 8.3 than that of B. natto. When this high producing activity of α-amylase (Amysup character) was transferred to a Marburg strain by the DNA from B. natto, about one-tenth of the transformants with Amysup character produced recipient type, i.e. Marburg type α-amylase and the other natto type α-amylase. In other words, the former acquired only high producing activity of α-amylase. This result suggests that a gene which regulates α-amylase synthesis participates in the α-amylase-producing system of B. subtilis and this regulator gene (amyR) is closely linked to the α-amylase structure gene. Reciprocal crossing also confirmed this assumption. The amyR gene is linked to the aro116 gene which has been reported to be the linked marker of α-amylase structure gene by S. Yuki.

Activity of the Escherichia coli cardiolipin synthase, encoded by cls, increased about 10-fold in the stationary growth phase, while other committedstep enzymes in phospholipid biosynthesis rather decreased. A null cls mutant lost viability to 10−4 of the wild-type cells during the prolonged incubation for 5 days. Cardiolipin was most stable among membrane phospholipids during the incubation. Accordingly, cardiolipin should play a role in survival of the cell and E. coli employs a sophisticated way to form cardiolipin according to need even under non-growing conditions.

The optic nerve of the bullfrog (Rana catesbeiana) was transected and the regeneration process was investigated. The nerve regeneration was confirmed by observing the extension of the axon and by examining the recovery of axonal transport. Following optic nerve transection, the rapid and transient increase of messenger RNA of α-tubulin gene was observed in the retina, but no specific change of actin messenger RNA was observed.

The 500 watts xenon lamp was used as a light source. The maximum luminance of the white object was set up to be 25,000 cd/m2 at 59′ visual angle and the maximum white background luminance was 2,200 cd/m2 at 10°.

The increment threshold versus intensity (t.v.i.) curve for rods was studied using Stiles’ two color threshold technique at 10° temporal retina on two normal observers. The results show that the t.v.i. curves for rods for both observers were shallower than the t.v.i. curves for rods which Stiles (1939) (1) described. The possibility that the t.v.i. curve for rods may consist of two brances was suggested. In order to examine the results with the Field Additivity Law, the t.v.i. curve for rods for a 430 nm test on a 480 nm background, and bichromatic mixtures of fields of 480 nm and 598 nm were measured at 10° temporal retina. In this field mixture experiment, the increment threshold for rods obeyed the Field Additivity Law.

Change of urate and ascorbate levels in retinae of rat left eyes after ophthalmic artery and optic nerve ligation was studied by reversed-phase high-performance liquid chromatography with electrochemical detection. The right eyes were used for sham operation. We found that uric acid levels in the retinae of the left eyes increased as a function of time after the operation, whereas those of the right eyes remained unchanged. In addition, we found that ascorbic acid levels in the retinae of the rat eyes decreased after the operation.

A study was made on the increment threshold versus intensity (t.v.i.) curves for a blue (480 nm) test on a yellow (590 nm) background at retinal eccentricities up to 10° on two normal observers. The observed t.v.i. curves at the extrafovea yielded four branches, i.e., for rods or π°(μ), the medium-wave sensitive mechanism or π4(μ), and the short-wave sensitive mechanisms or π1(μ) and π3(μ) respectively.

Six α-amylases (MAR, NAT, NA20, NA64, SAC and LIQ) produced by a transformable strain of Bacillus subtilis 6160, by a DNA donor strain of Bacillus natto IAM 1212, by representative transformants Bacillus subtilis NA20 and NA64, by Bacillus subtilis var. amylosacchariticus and Bacillus amyloliquefaciens F were compared with one another with regard to their immunological and chemical properties, including peptide mappings. Five α-amylases (MAR, NAT, NA20, NA64 and SAC) cross-reacted almost equally with rabbit anti α-amylase (SAC) serum but not with rabbit anti α-amylase (LIQ) serum, α-Amylases (MAR and NA64) contained about 9% of carbohydrate but not the others. The different indices calculated from the amino acid compositions showed not only structural similarity among the five α-amylases but also considerable difference between them. More than 80% of the tryptic peptide components from α-amylases (MAR and NA64) seemed to be identical and 4 or 5 specific peptides for each were detected. About 88% of the tryptic peptides from α-amylase (NAT) seemed to be in common with those from α-amylase (NA20). The latter contained more specific peptides than the former. The peptide maps of the five saccharifying type α-amylases (MAR, NAT, NA20, NA64 and SAC) showed that at least 12 peptides seemed to be common.

The spectral sensitivities for a 1°, 200-ms test flash on a white background were studied for up to 15° from the fovea on two normal observers as a basis for color perimetry. The results showed the usual three maxima of sensitivities, about 430 nm, 530 nm and 610 nm, at the fovea. However, the two peaks above 480 nm gradually decreased with increasing retinal eccentricity and at the 15° temporal retina, the curve showed one broad maximum in the region above 480 nm. That is, no R/G opponent color system could be detected. However, the peak at about 430 nm remained prominent even at 15° from the fovea.

The thread cells in the slime gland of Japanese hagfishes, Paramyxine atami and Eptatretus burgeri were studied by light and electron microscopy. The mature thread cells are large elements (180 times 80 mu) filled with an intricately coiled thread, approximately 2 mu in diameter. The protein nature of the thread has been confirmed by histochemical examination. In the initial stage of growth, the thread consists of a bundle of distinctly parallel filaments approximately 90-120 A in diameter and a centrally located tubular component approximately 230-260 A in diameter which occurs singly or occasionally as a double and triple structure. The developing thread displays thin filaments, approximately 30-60 A in diameter. The thin filaments are composed of fine fibrous structures, subfilaments, approximately 10-30 A in diameter. On the outer surface of the thread a coating is apparent, giving it a fluffy appearance. Polysomal clusters consisting of five or six ribosomes are predominant. Fine fibrous structures are also found among the threads; they seem to have a spatial relationship with the polysomes and resemble the subfilament constituents of the thin filaments. From these results, it may be suggested that the fine fibrous structures synthesized by polysomes, twist together and coalesce into a thread. The problem of the polysome size and the molecular weight of the fibrous protein synthesized is discussed.

An earthquake of JMA magnitude 6.5 (first event) hit Kumamoto Prefecture, Japan, at 21:26 JST, April 14, 2016. Subsequently, an earthquake of JMA magnitude 7.3 (second event) hit Kumamoto and Oita Prefectures at 01:46 JST, April 16, 2016. An out-of-service Kyushu Shinkansen train carrying no passengers traveling on elevated bridges was derailed by the first event. This was the third derailment caused by an earthquake in the history of the Japanese Shinkansen, after one caused by the 2004 Mid-Niigata Prefecture Earthquake and another triggered by the 2011 Tohoku Earthquake. To analyze the mechanism of this third derailment, it is crucial to evaluate the strong ground motion at the derailment site with high accuracy. For this study, temporary earthquake observations were first carried out at a location near the bridge site; these observations were conducted because although the JMA Kumamoto Station site and the derailment site are closely located, the ground response characteristics at these sites differ. Next, empirical site amplification and phase effects were evaluated based on the obtained observation records. Finally, seismic waveforms during the first event at the bridge site of interest were estimated based on the site-effect substitution method. The resulting estimated acceleration and velocity waveforms for the derailment site include much larger amplitudes than the waveforms recorded at the JMA Kumamoto and MLIT Kumamoto station sites. The reliability of these estimates is confirmed by the finding that the same methods reproduce strong ground motions at the MLIT Kumamoto Station site accurately. These estimated ground motions will be useful for reasonable safety assessment of anti-derailment devices on elevated railway bridges.

A method for producing a step-like change in a magnetic field has been developed. The apparatus used consists of two magnetically coupled superconducting magnets and two superconducting switches, and a sharp step-like transition of the magnetic field is produced in the magnets by operating the switches alternately. The magnets are in the persistent-current mode, and no power supply is required to keep the fields constant. A step-magnetic-field change of 2.1 T is obtained in a time interval of less than a second without any instability. The maximum rate of the change was estimated to be about 10 T/s, which is limited by the low normal resistance and slow thermal response of the superconducting switches. The change rate can easily be increased by more than two orders of magnitude when the characteristics of the switches are improved. The present method is especially useful for obtaining a large and rapid change in the strong magnetic field.