Immunochemical difference between cathepsin D and cathepsin E-like enzyme from rat spleen.

The immunological properties of acid proteinases from rat spleen, two types of cathepsin D and a cathepsin E-like enzyme, were examined. The rabbit antiserum was prepared against the major form of cathepsin D (cathepsin D-I) from rat spleen. The antiserum quantitatively precipitated the enzyme activity from the purified cathepsin D-I preparation. On immunodiffusion analysis, the antiserum showed an identical reaction with the minor form of cathepsin D (cathepsin D-II) from rat spleen. Immunoelectrophoresis showed that the precipitin line with cathepsin D-II ran somewhat faster to the anode than that with cathepsin D-I. The cathepsin E-like acid proteinaspe was neither precipitated nor inhibited by the antiserum to cathepsin D-I, indicating that the cathepsin E-like enzyme is different from cathepsin D. Immunological gel diffusion with the antiserum indicated that rat spleen cathepsin D was immunologically identical with cathepsin D obtained from rat brain, thymus, lungs, heart, liver, kidneys, and adrenals.

Effect of anticholinergic drug on long-term potentiation in rat hippocampal slices.

Long-term potentiation (LTP) was studied in CA1 neurons by tetanization of the Schaffer-commissural pathway in rat hippocampal slices. A brief tetanus (200 Hz for 1 s) caused an increase in amplitude of the population spike recorded from the CA1 area (typically about 200% of control), which lasted for more than 2 h. LTP was suppressed by perfusion of the muscarinic antagonist scopolamine (10(-5) M) from 5 min before to 15 min after the tetanus. If perfusion of the drug was begun after the tetanus, there was no affect on LTP. Scopolamine perfused without tetanus did not change the amplitude of the population spike. These results suggest that cholinergic system may affect the generation mechanisms of LTP.

A new method for application of horseradish peroxidase into a restricted area of the brain.

A new method for horseradish peroxidase (HRP) application into the very restricted area of the brain was developed. Crystalline HRP was packed into the tips of fine micropipettes having tip size of about 50 micrometer in outer diameter. The HRP micropipettes were stereotaxically implanted into the globus pallidus of rats. After survival period of 6 to 48 hours, brain was excised and HRP distribution was investigated following conventional procedures. The diffusion area of HRP at the application site was restricted within the limits of 200--250 micrometer in diameter completely inside the globus pallidus. It was known that the slowly solubilized crystalline HRP was taken up by the nerve terminals in the globus pallidus, and transported retrogradely to the cell bodies in the pars compacta of substantia nigra, indicating the existence of nigro-pallidal projections.

Sociomedical factors affecting participation in screening program on cerebro- and cardio- vascular disease.

In 1984 the Public Health Bureau of Nagoya City began a new health check-up program to encourage citizens aged 40 years to have a medical examination. The rate of participation was 46.6% greater than that of the previous program; however, at about 16%, it was still low. From the survey in Moriyama Ward following results were suggested. Housewives and female part-time workers who had not had a health examination during the previous year showed participation rates of 32.7% and 42.4%, respectively. It would seem that the low rate of participation in the health examination program conducted in the metropolitan area by the municipal Public Health Bureau is due to the existence of many medical care facilities, and the fact that 64.6% of men and 52.6% of women had undergone a medical examination in the year preceding the program. Total screening rates became 69.7% in males and 66.0% in females. Participation rates of 32.7% and 42.4% were achieved by unemployed women and by women with part-time employment, respectively, who had no experience of screening in the previous year. Total screening rates were 63.7% for the unemployed women and 82.1% for the part-time women.

Computed radiography utilizing laser-stimulated luminescence: detectability of simulated low-contrast radiographic objects.

Threshold contrasts of low-contrast objects with computed radiography (CR) images were compared with those of blue and green emitting screen-film systems by employing the 18-alternative forced choice (18-AFC) procedure. The dependence of the threshold contrast on the incident X-ray exposure and also the object size was studied. The results indicated that the threshold contrasts of CR system were comparable to those of blue and green screen-film systems and decreased with increasing object size, and increased with decreasing incident X-ray exposure. The increase in threshold contrasts was small when the relative incident exposure decreased from 1 to 1/4, and was large when incident exposure was decreased further.

Phantom evaluation of scatter and attenuation correction in thallium-201/technetium-99m acquisition in myocardial perfusion single-photon emission computed tomography.

PURPOSE: This phantom study was carried out to evaluate the usefulness of scatter correction combined with transmission-based attenuation correction in separate and simultaneous 201Tl/99mTc myocardial SPECT. METHODS: An anthropomorphic torso phantom was used in this study. We used the triple-energy-window (TEW) method for scatter correction and transmission computed tomography (TCT) images for attenuation correction. Images without corrections (UC) and images with corrections (SAC) for scatter and attenuation were reconstructed for the evaluation. RESULTS: The differences in defect size between 99mTc and 201Tl UC images led to interpretation errors in separate (separate protocol) and simultaneous dual-isotope studies (simultaneous protocol). These errors were more prominent in the infero-posterior wall in the simultaneous protocol. Improvement for overestimation in object size and underestimation in defect contrast was visually obtained, and increased contrast was also shown by the myocardium-to-defect count (MD) ratios on SAC images in the separate and simultaneous protocols. However, 201Tl SAC images in the simultaneous protocol still had less defect contrast than the corresponding 201Tl SAC images in the separate protocol. CONCLUSIONS: From the results of our phantom experiment, separate rest 201Tl/stress 99mTc-sestamibi acquisitions may be recommended in clinical practice. Further clinical and phantom studies will be needed to validate the method using scatter correction combined with transmission-based attenuation correction.

[Association between the interleukin 1B (C-31T) polymorphism and Helicobacter pylori infection in health checkup examinees].

PURPOSE: Associations between Helicobacter pylori (HP) infection and lifestyle factors have been demonstrated by several studies, but there are very few reports on links with host factors, especially concerning genetic polymorphisms for inhabitants of large city. The present investigation was conducted to determine the HP infection rate with reference to the Interleukin-1 beta gene (IL-1B) polymorphism and assess the interactions with smoking reported for outpatients. METHOD: The subjects were 468 participants in a health-check-up program of law of health for aged conducted by Nagoya Nishi Health Center. The participants were asked to permit use of their residual blood drawn during a health-check-up program and written informed consent was obtained for gene polymorphism tests. Data on smoking habit were obtained by self-administered questionnaire. The IL-1B C-to-T polymorphism at -31 was genotyped by PCR-CTPP (polymerase chain reaction with confronting two-pair primers) and an anti-HP IgG antibody test was used for detecting HP infection. Differences in values were assessed by a chi 2 test. An unconditional logistic model was applied for estimating odds ratios with the computer program STATA Version 6. This study was approved by the Ethical Committee at Aichi Cancer Center in 2000 before it was commenced (Ethical Committee Approval Number 11-12) RESULTS: The HP infection rate was 52.6% (61/116) for the C/C genotype of IL-1B-31, 48.6% (89/183) for the C/T, and 63.2% (103/163) for T/T; the difference were not statistically significant. However, when non-current smokers were excluded, the rate were 47.8% (11/23), 52.9% (18/34), and 72.7% (16/22), respectively, indicating the T/T genotype to have a higher infection rate. The age-sex-smoking adjusted odds ratio (OR) relative to the C/C genotype were 0.97 (95% confidence interval, 0.59-1.57) for the T/C genotype and 1.73 (1.04-2.87) for the T/T genotype. Among current and former smokers the age-sex adjusted OR were 1.68 (0.50-5.71) for the T/C genotype and 5.29 (1.11-25.1) for the T/T genotype, suggesting a effect of this polymorphism prominent in smokers. CONCLUSION: An association between the IL-1B-31 polymorphism and persistent HP infection was observed for inhabitants with a smoking habit, indicating that this polymorphism is one genetic trait conferring an increased likelihood of persistent HP infection.

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.

2-amino-5-phosphonovaleric acid blocks induction of an epileptiform discharge following a brief hypoxic episode in the hippocampal slices prepared from dietary Mg-deficient mouse.

The effects of a brief hypoxic episode on synaptic activity in the CA1 region of the hippocampus were studied using the in vitro slice prepared from dietary Mg-deficient mouse. After 5 min of hypoxia, the synaptically evoked population spike was increased in amplitude and developed to an epileptiform discharge. The induction of the epileptiform discharge following hypoxia was prevented in the presence of DL-2-amino-5-phosphonovaleric acid (APV), a N-methyl-D-aspartate (NMDA) receptor antagonist, at a concentration of 30-50 microM. The post-hypoxic epileptiform discharge, however, was reduced but not blocked completely by the application of APV (concentration up to 100 microM). These results show that a brief hypoxic episode induces APV-sensitive epileptiform activity in the CA1 region of dietary Mg-deficient mouse, suggesting the involvement of NMDA receptors in post-hypoxic changes of synaptic transmission in the hippocampus.

[Protective effect of suloctidil against cerebral hypoxia (author's transl)].

The protective effect of suloctidil [MY 103, erythro-1-(4-isopropylthiophenyl)-2-n-octylaminopropanol] against cerebral hypoxia was investigated in terms of survival time under normobaric hypoxia (96% N2 + 4% O2 gas mixture) in mice and evoked potential in prepyriform cortex of guinea pig in vitro. In a hypoxic condition, the pretreatment with MY 103 (3.0-12.5 mg/kg, i.p.) resulted in a marked and dose-dependent prolongation of survival time of mice without any adverse effect. The prolongation of survival time was statistically significant at doses over 6.3 mg/kg. The amplitude of evoked potential to electrical stimulation of olfactory tract in the brain slice was consistently and reversibly suppressed by the perfusion with low oxygen Ringer's solution containing about 45% O2 or normal Ringer's solution. This amplitude reduction after 6 minutes exposure to hypoxic perfusate, was consistently reproducible in the same preparation. Preincubation of brain slices in normal Ringer's solution containing 10(-7)-10(-6) g/ml MY 103 for 15 min, however, effectively prevented the hypoxic reduction of the amplitude of evoked potentials to 30-50% of the control experiments. The above data indicate that MY 103 exerts its anti-anoxic effect presumably through modified metabolic processes such as active oxygen or glucose uptake by the brain.

[Effect of suloctidil on the reduced evoked potentials in brain slices in low glucose perfusate].

Effects of suloctidil [erythro-1-(4-isopropylthiophenyl)-2-n-octylaminopropanol] on reduced evoked potentials in low glucose perfusate were studied using guinea-pig prepyriform cortex in vitro. The amplitude of evoked potential to paired electrical stimulation of the olfactory tract in cerebral slices was consistently and reversibly suppressed by the perfusion with low glucose (3 mM)-Krebs-Ringer's solution. The amplitude reduction after 10 min exposure to the perfusate was fairly reproducible in the same preparation. The amplitude reduction was always accompanied with significant facilitation of "postactivation potentiation", estimated from the size of the first and second response to paired stimulation. Perfusion of brain slices with low glucose Krebs-Ringer's solution containing 10(-7)-10(-6) g/ml suloctidil for 10 min, however, effectively prevented the reduction of amplitude of evoked potentials to below 30-50% of the control value. The facilitation of postactivation potentiation under the low glucose condition was also suppressed by the drug. Dihydroergotoxin, 3 X 10(-6) g/ml, also showed a comparable effect as 10(-6) g/ml suloctidil. These data suggest that suloctidil possesses a brain energy economizing effect such as activation of oxygen, glucose uptake or production of high energy substance.

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.

Detection of subtle microcalcifications: comparison of computed radiography and screen-film mammography.

Observer performance tests were conducted to compare the effects of image-processing technique (unsharp mask filtering) on the diagnostic accuracy of computed radiography (CR) with storage phosphors in the detection of microcalcifications. Comparison of detectability of microcalcifications with CR and with screen-film mammography was also performed. Clusters of microcalcifications (125-250 microns in diameter) were randomly superimposed on human breast specimens. Observer performance tests were carried out with receiver operating characteristic (ROC) analysis. The area under the ROC curve and the cumulative true-positive-localization fraction were both used as indexes of performance. Observer performance experiments with nine observers indicated that the two types of screen-film images used provided higher detectability than the CR images. The detectability of microcalcifications on the CR images improved slightly with use of the unsharp masking technique. However, no statistically significant difference was found between processed and unprocessed CR images, and detectability still did not reach the level achieved with the screen-film images.

Smoking habit and interleukin lB C-31T polymorphism.

Recent studies suggest that smoking habit may relate to genetic traits. This study examines the association with a polymorphism (C-31T) of interleukin 1B (IL-1B), which encodes IL-1beta, a multifunctional pro-inflammatory cytokine. Since the T allele makes a TATA box, the allele is thought to be responsible for a higher potency of IL-1B expression, indicating that individuals harboring the T allele are prone to inflammation. The study subjects were two different populations; 241 non-cancer outpatients (118 males and 123 females) at a cancer hospital and 462 examinees (127 males and 335 females) of a health checkup program provided by a local government. Current smokers were 36.4% for the male outpatients, 9.8% for the female outpatients, 38.6% for the male examinees, and 5.6% for the female examinees. The sex-age-adjusted odds ratios of current smokers were calculated for the genotypes with the T allele relative to the CC genotype by an unconditional logistic model. The estimate was 0.45 (95% confidence interval, 0.21-0.97) for the outpatients, and 0.83 (0.42-1.61) for the examinees. Although not significant for the examinees, the observed associations suggest that this polymorphism may influence smoking behavior through an inflammatory response of the respiratory tract to cigarette smoke.

[Preliminary clinical applications of asymmetric screen-film systems in chest radiography].

The physical imaging properties of asymmetric systems and a conventional system were evaluated by measuring characteristic curves, resolution properties and noise Wiener spectra. The potential clinical application of asymmetric screen-film systems was studied by evaluating the visibility of the anatomical structures and various types of abnormalities in comparison with those of a conventional screen-film system. The asymmetric systems showed a wider dynamic range than the conventional system. The resolution properties of asymmetric systems depend on the combination of front and back screens used. Chest radiographs obtained with asymmetric systems improved the visibility of the mediastinal area. The visibility of the lung field in the asymmetric systems was slightly inferior to that with the conventional system when the same tube potential was used. However, the image quality and visibility of various abnormalities showed greater improvement with the asymmetric systems when a lower tube potential was applied. We conclude that the selection of radiographic techniques and combination of screens are important for the clinical use of asymmetric systems.