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Biomedical subjects

S Nojima

Publications and source records attributed to S Nojima.

At least 55 records · Page 3Linked to original sources
PubMed1991

Radioimmunoassay for platelet-activating factor.

A radioimmunoassay (RIA) for measurement of platelet-activating factor (PAF) was developed. At a final antiserum dilution of 1:640, the lowest detection limit of PAF was 0.1 pmol (50 pg). The standard curve obtained was suitable for measurement of PAF in amounts ranging from 0.1 pmol to 30 pmol. The antiserum showed high specificity. Cross-reaction for lysoPAF, lysophosphatidylcholine and long-chain phosphatidylcholines was very low (less than 0.025%). 1-Palmitoyl-2-acetyl-sn-glycero-3-phosphocholine cross-reacted slightly (6.25%). PAF exogenously added to macrophage suspensions was quantitatively determined by RIA after solvent extraction and high-performance liquid chromatographic separation. RIA was also used to estimate PAF formation after stimulation of rabbit alveolar macrophages in suspension with calcium ionophore A23187.

Animals↗
PubMed1991

Biological response of guinea pig peritoneal macrophages to platelet-activating factor.

We investigated the effects of platelet-activating factor (PAF) on guinea pig peritoneal macrophages. Specific and high-affinity binding sites for PAF were detected on guinea pig peritoneal macrophages. Scatchard analysis of PAF binding revealed high affinity binding sites (7.9 x 10(4)/cell) with a dissociation constant of 2.3 x 10(-10) M. When treated with 10(-9)-10(-5) M PAF, guinea pig peritoneal macrophages released hydrogen peroxide into the medium in a time-dependent manner. The release reaction upon stimulation with 10(-5) M PAF reached a plateau within 30 min and the extent of release was twice as high as that when stimulated by N-formyl-L-methionyl-leucyl-L-phenylalanine (fMLP; 2 microM)-treated cells. Neither lysoPAF nor the PAF enantiomer was effective. PAF-induced H2O2 release was inhibited specifically by PAF antagonists, suggesting that PAF activated macrophages through binding to specific sites. Lysosomal enzyme (N-acetyl-beta-D-glucosaminidase) was released from guinea pig peritoneal macrophages upon treatment with 10(-5) M PAF for 60 min. Guinea pig peritoneal macrophages were treated with PAF for 8 hr and the conditioned medium was examined for cytokines. The medium exhibited cytocidal activity against mouse fibroblast L929 cells [tumor necrosis factor (TNF) activity], and this activity was comparable to that detected after treatment of cells with the bacterial lipopolysaccharide (LPS). Furthermore, the same conditioned medium also showed colony-stimulating factor (CSF) activity. Generation of these cytokines was stereospecific. Our findings suggest that PAF is a unique macrophage activator that potentiates both respiratory burst/lysosomal enzyme release (early-phase response) and monokine production/glucose consumption (late-phase response).

Animals↗
PubMed1991

Antibodies to synthetic platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) analogues with substituents at the sn-2 position.

We obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentadecyl)-2-O-acetyl-sn-glycero-3- phosphocholine(acetyl-CPGPC), 1-O-(15'-carboxypentadecyl)-2-O-N,N-dimethylcarbamoyl-sn-glycero-3 - phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O-(15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-pho sphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl much greater than dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethylcarbamoyl greater than acetyl much greater than butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC-BSA; and butylcarbamoyl greater than dimethylcarbamoyl greater than acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these antibodies. Anti-dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG inhibited a PAF-induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG.

Animals↗
PubMed1991

[Effects of video games on visual function in children].

Health effects of video games were examined with a questionnaire survey and an experimental study. In the survey, age, sex, playing time with video games in a day, viewing distance between the eyes and the television screen, subjective symptoms of eye strain, and changes of visual acuity were investigated in 2,034 male and 2,321 female primary school children. In the experimental study, the visual loads of a one-hour word processing task and a video game were compared with various indices, such as critical flicker fusion, near point distance, blinking counts, eyeball movement, pupillary reflex and subjective fatigue symptoms in ten healthy male college students. The results of this study were as follows: 1) The viewing distance between the eyes and the TV screen was shorter for video games than for watching TV programs. 2) The rate of complaints related to eye strain in children who played video games over 120 min per day was significantly higher than that of other children. 3) The eye movements during video games were more rapid and frequent than those during conventional VDT work. 4) A decrease in CFF value and an extension of near point distance and an increase in subjective fatigue symptoms were demonstrated for both the word processing task and the video game. No significant differences were observed between the above two experimental conditions. This suggests that the visual loads for video games are similar to those for VDT work. These findings suggest that regulating the playing times to 60 minutes or less per day is necessary to prevent negative health effects in children.

Asthenopia↗
PubMed1991

Lysophospholipase L1 from Escherichia coli K-12 overproducer.

After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S. (1985) J. Biochem, 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa.

A (H+ + K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation. The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na+ + K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase). The (H+ + K+)-ATPase had a pH optimum of 7.4 and was stimulated by Tl+, K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM, respectively, at this pH. On the other hand, monovalent cations such as Na+, Li+ and (CH3)4N+ as well as divalent cations such as Cu2+, Ca2+, Ba2+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently. Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H+ + K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed a major band (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-32P]-ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H+ + K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

Adenosine Triphosphatases↗
PubMed1989

ESR study on synthetic glyceroglycolipid liposomal membranes.

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmitoyl[beta-cellobiosyl-(1'---3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37 degrees C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-6-DPPA in the Cel-DAG liposomes at 37 degrees C showed broad resonance lines with the central peak being the highest, while that at 60 degrees gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37 degrees C but not above Tc. These results suggest that below Tc the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

Alternative usage of different poly(A) addition signals for two major species of mRNA encoding human aromatase P-450.

Two cDNA clones for human placental aromatase P-450 (P-450AROM) have been isolated and sequenced. The insert of one clone (2894 bp) contains an open reading frame encoding a protein consisting of 503 amino acid residues together with a 49 bp 5'-untranslated stretch and a 1336 bp 3'-noncoding region to which a poly(A) tract is attached. Three potential poly(A) addition signals are detected in this 3'-noncoding region. The other clone contains a shorter cDNA insert, the nucleotide sequence of which overlaps with most of the sequence of the longer cDNA insert (nucleotides 36-2355) except for one nucleotide substitution. The 3'-noncoding region of this shorter cDNA is only 846 bp in length but a poly(A) tract is also attached to its 3'-terminus. Northern blot analysis of human placental RNA reveals the presence of two major mRNA species of 3.4 and 2.9 kb when probes excised from the overlapping region of these two cDNAs are employed. The 2.9 kb mRNA is not detected, however, when a fragment of the non-overlapping region of the longer cDNA is used as a probe. It is therefore concluded that the two major species of P-450AROM mRNA are formed as a consequence of alternative processing of precursor mRNA(s).

Amino Acid Sequence↗
PubMed1988

A novel bioaction of PAF: induction of microbicidal activity in guinea pig bone marrow cells.

When guinea pig bone marrow cells were incubated in the presence of 10(-8) to 10(-6) M platelet activating factor (PAF) for 24 to 72 hr, microbicidal activity against Candida parapsilosis of cells was augmented. This augmentation was inhibited by PAF-specific antagonists, CV6209 or FR900452. PAF-specific binding sites with a high affinity were found on guinea pig bone marrow cells. Carrageenan or 2-chloroadenosine, reagents known to be preferentially cytotoxic to macrophages, abolished the microbicidal activity of PAF-treated bone marrow cells. Macrophages prepared from the peritoneal cavity, however, acquired no appreciable microbicidal action by treatment with PAF. These observations suggest that PAF may affect a class of guinea pig bone marrow cells through binding to receptors specific to PAF, resulting in activation and/or induction of differentiation of monocyte-macrophage lineage cells.

2-Chloroadenosine↗
PubMed1988

Lack of protein-mediated alpha-tocopherol transfer between membranes in the cytoplasm of ascites hepatomas.

Transfer-stimulating activity for alpha-tocopherol and the concentration of alpha-tocopherol and peroxidized lipids in rat ascites hepatoma cells were compared with those from normal and regenerating liver. The ability of supernatants from ascites hepatomas (AH-13, AH-60C, AH-109A) to enhance the transfer of alpha-tocopherol was much lower than that from normal livers. The alpha-tocopherol per mg protein of supernatant from ascites hepatomas was lower than that from normal liver. Regenerating liver showed almost the same values as normal liver in activity to stimulate the transfer of alpha-tocopherol and alpha-tocopherol content of the supernatant. By gel filtration, about 60% of alpha-tocopherol in the supernatant of normal liver was detected in the fractions containing the 30 K protein, which stimulates transfer of alpha-tocopherol between membranes, whereas no significant amount of alpha-tocopherol was detected in 30 K protein fractions of AH-60C supernatant. Little stimulating activity for alpha-tocopherol transfer was detected in AH-60C, AH-109A and AH-13. All ascites hepatomas tested contained less arachidonic acid and docosahexaenoic acid than normal and regenerating liver. An absorption peak with maximum intensity at 233 nm, which is due to conjugated dienes, was observed in UV-absorption spectra of ascites hepatoma total lipids, indicating that peroxidized lipids accumulate in these cells. With normal and regenerating liver, no significant peak due to conjugated dienes was detected.

Animals↗
PubMed1988

Hydrolysis of platelet activating factor and its methylated analogs by acetylhydrolases.

We examined the substrate specificity of PAF-degrading enzymes from various sources using platelet activating factor (PAF) and its synthetic analogs. The results were as follows: 1) Tissue-originated acetylhydrolases, such as rat kidney soluble enzyme, deacetylated 1S-methyl-1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (1S-Me-PAF) slightly more rapidly than PAF, whereas plasma acetylhydrolase hydrolyzed PAF more effectively than 1S-Me-PAF. 2) Rat polymorphonuclear leukocytes, monocytes, and lymphocytes homogenates showed an appreciable acetylhydrolase activity, the substrate specificity of which resembled that of the plasma enzyme. 3) Pleural exudates in an experimental pleurisy induced in rats by carrageenan contained an acetylhydrolase activity, the properties of which were similar to those of the plasma enzyme. 4) An extracellular phospholipase A2 activity, which was also observed in the pleural exudate and required Ca2+ ion for maximum activity, seemed not to participate in the deacetylation of PAF, since addition of EDTA did not affect the PAF deacetylation catalyzed by the pleural exudate. These findings indicate that the inactivation reaction of PAF present in the extracellular space is mainly catalyzed by plasma acetylhydrolase, which yields lysoPAF.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗