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Surfactant treatment in infants with respiratory distress syndrome (RDS) has decreased neonatal mortality. With the advent of this therapy, it has become important to predict accurately the fetal lung maturity of a fetus before delivery. We evaluated the stable microbubble test (SMT), surfactant protein-A (SP-A) and hepatocyte growth factor (HGF) in amniotic fluid as predicting markers for RDS. Of 55 amniotic fluid samples obtained by amniocentesis from women less than 37 weeks pregnant, the SMT values were as follows: sensitivity 76.5%, specificity 84.2%, positive predictive value 68.4%, negative predictive value 88.9% and overall accuracy 81.8%. For SP-A, the values were 88.2%, 65.8%, 53.6%, 92.6% and 72.7%, respectively. If we used both SMT and SP-A, we could diagnose with 100% accuracy that a case with measurements of SMT > or = 2 and SP-A > or = 420 ng/ml would not complicate with RDS (24/24). However, the RDS diagnostic accuracy of HGF does not equal to those of SMT and SP-A levels. We concluded that the rapidity, simplicity and reliability of SMT was very useful during 24-36 weeks of gestation as a bedside procedure to predict fetuses likely to develop RDS. We also noted the additive effect of SP-A in improving the accuracy of lung maturity diagnosis.

Several specific assays have been developed for the measurement of etoposide in biological fluids. As large samples are required for high sensitivity, these systems are not appropriate for a pediatric practice. In the present study, however, an improved method for the determination of serum levels of the anticancer drug etoposide was developed, using high-performance liquid chromatography with fixed-wavelength ultraviolet detection. Etoposide was extracted from serum using dichloromethane. The efficiency of extraction from serum was 85.7 +/- 7.7% for etoposide and 81.1 +/- 8.4% for diphenylhydantoin, the internal standard. The serum concentrations of etoposide were measured in 0.2-ml serum samples. The lower limit of detection was 50 ng/ml. Each measurement was completed within 5 min. The linear quantitation range for etoposide was 0.05-50 microg/ml. This assay presents an alternative method for routine measurement of serum levels of etoposide in the pediatric oncology setting.

We observed the distribution of CD56+ epithelial cells in the pancreatic duct system using 25 fetal, one infantile, 3 normal adult, 4 diabetic, and 8 chronically inflamed pancreatic tissue samples. In the early stage of gestation (12 to 17 weeks), CD56+ cells were commonly seen in the immature tubular structures. They were often continuous to pancreatic islets, and their distribution was similar to that of synaptophysin (Syn)+ cells, suggesting that they are precursors of islet neogenesis. Their number decreased in proportion to gestational age. Instead, from 24 weeks of gestation, luminal cell clusters that were common in interlobular ducts revealed CD56+. These cell clusters were unrelated to islet neogenesis and Syn expression. Similar CD56+ luminal cell clusters were also observed in cases of chronic pancreatitis, whereas they were scarce in normal adult and diabetic tissues. CD56+ cells were also occasionally seen in intralobular ducts, intercalated ducts, and centroacinar cells in cases of chronic pancreatitis. We conclude that there are two types of CD56+ epithelial cells in the pancreatic duct system: CD56+ endocrine cells are numerous during the early stage of gestation, when islet neogenesis appears, while CD56+ luminal cells may represent developmental and regenerative changes of pancreatic ducts.

With the purpose to clarified the mode of localization and mechanisms of activation of ATPase in the mitochondrial membrane, analyses were made on the properties of mitochondrial ATPase from the structural and functional aspects. The activation of ATPase by DNP and Mg++ and the oligomycin sensitivity were investigated in a series of inner membrane fragment samples obtained by ultrasonic irradiation and those samples obtained in the processes of isolation and purification of ATPase from rat liver mitochondria and beef heart mitochondria in parallel with electron microscope observations. As a result it has been found that the membrane fragments obtained from rat liver and beef heart mitochondria by ultrasonication exhibited high respiratory activity and unmasked ATPase activity which was charac· terized by remarkable stimulation by Mg++ and inhibition by oligomycin and azide. Therefore, mitochondrial ATPase seems to be bound fairly closely to the inner mitochondrial membrane. In the membrane fragments prepared by ultrasonication of intact mitochondria, ATPase activity was stimulated by DNP, but in the supernatant fractions was not. On the other hand, the supernatant fraction obtained from BHM and inner membrane fragments by severe sonication exhibits a marked ATPase activity and the activity incresed in each step of the purification on the treatments with acid, protamine and heat. Especially in the case of membrane fragments the protamine treatment can be omitted. Electron microscope observation of the fractions in each step of the purification proved the head pieces to be ATPase. The ATPase activity of solubilized head pieces is insensitive to oligo. mycin and coincides with the soluble ATPase of PULLMAN etat. (8) in the points of its cold labile property and optimum pH, but it shown no accele. ration of ATPase activity by DNP.

For the purpo3e to determine exactly what stage of cell specialization the DNA level of erythroid cell nuclei begins to decline, the author observed the DNA level of erythroblasts in mitosis by microspectrophotometry and the DNA synthesis by flash labeling with H3-thymidine. The cell samples were obtained from the bone marrow of normal, blood-depleted and phenylhydrazine-treated animals, and the anemic animals received a mass red cell transfusion, all the animals being injected with colchicine 4 hours before obtaining the bone marrow sample. DNA level was measured on the smeared cells stained by Feulgen reaction and DNA synthesis by autoradiography on the smeared cells. Besides these, chromosome number was observed on the anemic rat erythroblasts at metaphase by air dry method. The observations indicated that the DNA level begins to decrease at polychromatic stage being accompanied by a decrease in TDH3-incorporation into DNA, reaching minimum level at orthochromatic cell both in DNA contents and synthesis. Chromosome numbers of erythroblasts of rat were irregular being distri buted between 42 to 20. The data have suggested that the DNA level of erythroblasts decreases only in the later stages of cell specialization, and at polychromatic stage the chromosome number may also decrease in rabbit at polychromatic stage by the cell division with an incomplete DNA replication. The high DNA level of the erythroblasts of rabbit, in severe anemia where most of the cells are denucleated at polychromatic and late basophilic stages, has been discussed from the view point of the insufficient DNA replication at polychromatic stages.

Chromatography on Sephadex G-200 was performed with the soluble fraction of homogenated rabbit liver, which was extracted with 0.1 M phosphate buffer containing 0.1 M NaCl. and the influences of autolysis on the soluble fraction of liver were also examined. The soluble fraction of liver was different from serum in molecular weight, in electrophoretic character and in components with sedimentation coefficients. The soluble fraction of liver was stable under the influence of Mg and K ions, and rather unstable in the presence of Na ions. Serum was fractionated in three main peaks. The soluble fraction of liver was fractionated in a similar pattern as of serum, but the first peak contained nucleic acid and lipoprotein. The second contained albumin. 32p radioactivity peaks of the stored sample appeared with change in patterns by autolysis from the original, and were observed wide based and continuous figures in retarded peaks. The correlations with the first peak and retarded peaks were represented by the analysis of phosphorus compounds and electrophoresis. In lipid analysis, both diglyceride and monoglyceride gradually decreased, and phospholipid pattern was observed to increase in retarded peaks by autolysis. Lipoprotein or lipid-albumin complex was gradually converted to smaller molecular weight compounds, and appeared in retarded peaks.

To analyze the appearance of three forms of hepatitis B antigen-associated particles (HB Ag particles) and antigen-antibody (Ag-Ab) complexes in the sera of patients with various liver diseases, electron microscopic observations with the combinations of a variety of immunological assays were made at first on the HB Ag and Ab mixed in vitro in various ratios, and then on the samples from the sera of each patient. The number of patients observed were 64 in total, which consisted of various types of hepatitis, Hodgkin's disease, Down's syndrome and an asymptomatic carrier. For the detection of HB Ag-Ab complexes a modified method of ALMEIDA was used, and for the isolation of large HB Ag particles (Dane particles) DANE'S method was employed. Electron microscopy proved to be a useful method for detecting HB Ag and the Ag.Ab complexes when the ratio of HB Ag to Ab was in the equivalence. Large aggregates of Ag-Ab complexes were frequently observed in the attacks of acute hepatitis and the recrudescences-of chronic aggressive hepatitis. The aggregates were also observed in fulminant hepatitis but the ratio of HB Ag to Ab was different from each other among 3 cases examined. The large HB Ag particles were not observed in more than half of the cases in the attacks of acute hepatitis, but appeared in the major. ity of cases in chronic aggressive hepatitis, even massively during the period with transiently elevated levels of serum glutamic pyruvic transaminase. A few large particles were also found in sera of an asymptomatic carrier, Hodgkin's disease, and Down's syndrome.

The disappearance of nucleolus has been traced in the rat erythroid cells in relation with the cell specialization under varying conditions, i. e. in anemia with or without treatment by bromouracil and aminopterin. To make the findings more reliable the observations have been made on tissue section as well as on the smeared samples as the nucleolus becomes often indistinct in smeared cell. The results indicate that under anemic condition nucleolus is lost by the late basoplilic stage. Treatment with bromouracil retained the nucleoli and cytoplasmic basophilicity till later stage of cell specialization suggesting some similar mechanism of RNA disintegration both in nucleolus and cytoplasm.

The site of localization of TCA cycle dehydrogenases in mitochondria has been investigated by observing the dehydrogenase activities and fine structure of the fractionated samples after freezing and thawing or sonication of beef heart and rat liver mitochondria. 1. In the sonicated mitochondria, activities of malic and isocitric dehydrogenases were highest in the supernatant fraction centrifuged at 198,000 x g for 60 minutes, while the specific activity of a-ketoglutaric dehydrogenase was higher in the fluffy or residue fraction. The distribution of the activity of pyruvic dehydrogenase was similar to that of a-ketoglutaric dehydrogenase. 2. In a sucrose density gradient fractionation of the fluffy fraction obtained by centifugation of sonicated mitochondria at 198, 000 x g for 60 minutes, the activities of malic and pyruvic dehydrogenase were observed in the top (or low density) layer in the form of fine particles, while that of a-ketoglutaric dehydrogenase was observed in the middle (or medium density) layers in the form of aggregates of fine particles and membranous fragments. 3. In the samples fractionated after freezing and thawing of mitochondria, which were considered to be a relatively mild disruption, the specific activity of a-ketoglutaric dehydrogenase was higher in the residue (submitochondria) fraction than that in the supernatant fraction (centrifuged at 144,000 x g, 30 minutes), and the activity of malic dehydrogenase still remained significantly high in the residue fraction. 4. It was deduced that the TCA cycle dehydrogenases could be localized in the matrix of the mitochondria by a loose binding to the inner membrane.

We present here a reliable and sensitive method for the determination of acidic opines such as meso-alanopine, beta-alanopine, tauropine and strombine in biological samples. Interfering primary amino acids were eliminated by reaction with o-phthalaldehyde, and the derivatized compounds were passed through Sep-Pak Plus PS-1 cartridges. The acidic opines were recovered by flushing the cartridges with water, then determined by high performance liquid chromatography after a second derivatization with phenylisothiocyanate. All 4 acidic opines were detected within 30 min. This method ensured good separation and guaranteed almost full recovery of all acidic opines. This method was applied to analyze opines in marine animals and to test whether opines are metabolized in the livers of the rat and fish.

In this study, sex determination using polymerase chain reaction (PCR) on tooth material was evaluated from the viewpoint of forensic medicine. The sensitivity of PCR for detection of the Y chromosome-specific alphoid repeat sequence and the X chromosome-specific alphoid repeat sequence was 0.5 pg of genomic DNA. Sex could be determined by PCR of DNA extracted from the pulp of 16 freshly extracted permanent teeth and dentine including the surface of the pulp cavity of 6 freshly extracted milk teeth. Sex could be determined using the pulp in all 20 teeth (10 male and 10 female) preserved at room temperature for 22 years. For the pulp of teeth stored in sea water, the sex could be determined in all 8 teeth immersed for 1 week and in 5 of 6 teeth immersed for 4 weeks. In the remaining 1 tooth, in which sex determination based on the pulp failed, the sex could be determined correctly when DNA extracted from the tooth hard tissue was examined. For teeth stored in soil, the sex could be determined accurately in all 8 teeth buried for 1 week, 7 of 8 teeth buried for 4 weeks, and in all 6 teeth buried for 8 weeks. When teeth were heated for 30 min, sex determination from the pulp was possible in all teeth heated to 100, 150, and 200 degrees C, and even in some teeth heated to 250 degrees C. When this method was applied to actual forensic cases, the sex of a mummified body estimated to have been discovered half a year to 1 year after death could be determined readily by examination of the dental pulp. In the skeletons of 2 bodies placed under water for approximately 1 year and approximately 11 years and 7 months, pulp tissues had been dissolved and lost, but sex determination was possible using DNA extracted from hard dental tissues. These results indicate that this method is useful in forensic practices for sex determination based on teeth samples.

While heat shock protein (HSP) 72 is known as a stress protein, there have been no reports of HSP 72 expression in patients who have undergone surgery for congenital heart disease. Fourteen patients (7 males and 7 females) who had undergone surgery for congenital heart disease were studied. The ages of the patients ranged from 2 months to 43 years old (mean 6.5 +/- 10.8 years old; median 3.0 years old). The diagnoses were Tetralogy of Fallot in seven, pulmonary atresia with ventricular septal defect (VSD) in three, complex anomalies in three, and VSD in one patient. Histological study and HSP analysis using Western blots and immunostaining with anti-HSP 72 monoclonal antibody were performed for right ventricular muscle samples resected during the surgery. The histological findings showed hypertrophic changes of ventricular cardiomyocytes in all samples studied. Western blots detected HSP 72 expression of various degrees in all specimens. Immunostaining using monoclonal antibody against HSP 72 showed that the protein was present in the nuclei and cytoplasm of cardiomyocytes. In conclusion, although it is difficult to determine the cause of the "stress" that triggers HSP 72 expression in cardiomyocytes, low O2 saturation and pressure overload might act as a "stress", and the only common factor that induced HSP 72 in every sample was hypertrophy.

The effect of Blastocystis hominis (B. hominis) in both immunocompetent and immunocompromised subjects has been the subject of debate in recent years, mostly in response to its unknown pathogenicity and frequency of occurrence. We performed a non-randomised, open labelled, single institute study in our hospital in order to investigate the clinical significance and frequency of B. hominis in patients suffering from hematological malignancy (HM) who displayed symptoms of gastrointestinal diseases during the period of chemotherapy-induced neutropenia. The presence and potential role of other intestinal inclusive of parasites were also studied. At least 3 stool samples from each of 206 HM patients with gastrointestinal complaints (the HM group) were studied. These were compared with stool samples from a control group of 200 patients without HM who were also suffering from gastrointestinal complaints. Samples were studied with saline-lugol, formalin-ether, and trichome staining methods. Groups were comparable in terms of gender, age and type of gastrointestinal complaints. In the HM group, the most common parasite was B. hominis. In this group, 23 patients (13%) had B. hominis, while in the control group only 2 patients (1%) had B. hominis. This difference was statistically significant (P < 0.05). Symptoms were non-specific for B. hominis or other parasites in the HM group. The predominant symptoms in both groups were abdominal pain (87-89.5%), diarrhea (70-89.5%), and flatulence (74-68.4%). Although all patients with HM were symptom-free at the end of treatment with oral metranidazol (1,500 mg per day for 10 days) 2 patients with HM had positive stool samples containing an insignificant number of parasites (< 5 cells per field). In conclusion, it appears that B. hominis is not rare and should be considered in patients with HM who have gastrointestinal complaints while being treated with chemotherapy. Furthermore, metranidazol appears to be effective in treating B. hominis infection.

A study was conducted to analyze the distribution and diameter of muscle fiber types in samples of the medial paravertebral lumbar muscle, i.e., multifidus muscle, obtained from 76 patients who underwent surgery for disc herniation. The samples were compared with 41 control samples of corresponding muscle tissue taken from 41 young healthy subjects who had died a sudden death. Histochemical analysis of fibers associated with myofibrillar adenosine triphosphatase (ATPase) revealed the presence of Type I fibers (slow-twitch fibers) and of Type IIA and IIB fibers (fast-twitch fibers) in both the experimental and control samples. The respective percentage of muscle fibers was calculated and their diameters were measured. Type I fibers predominated in both groups and were significantly larger in diameter than Type IIA and IIB fibers. Both fast-twitch fiber types were distributed in almost equal proportions in the healthy women. In the healthy men, Type IIA fibers prevailed. In the healthy females, the percentage of Type I fibers was found to be slightly higher than in the males, but the diameter of all fiber types was respectively smaller. In the females who had undergone surgery, Type I fibers were significantly larger in diameter than those of the healthy subjects. On the other hand, the diameters of all muscle fiber types were significantly larger, and the percentage of both fast-twitch fibers were Idwer in the samples from men who underwent surgery, as compared to the healthy tissue samples. The morphometric changes in the multifidus muscle at the level of the protruded disc observed by the histochemical method for demonstration of myofibrillar ATPase could not be related to the compressed nerve root in the majority of cases in our study.

A study was performed to verify that the prevalence of alcohol abuse and dependence in Formosan aborigines differs from that of Taiwanese (Chinese Han people), using analysis of aldehyde dehydrogenase (ALDH) isozymes and flush patterns on randomly sampled 70 Atayal, 66 Paiwan, 61 Yami and 94 Taiwanese subjects were studied. The activity of an isomer of ALDH having a low Km (ALDH-I) in hair roots was analysed by isoelectric focusing assay. The subjective experience of flushing response after alcohol ingestion was assessed. Results showed that the rate of ALDH-I deficiency in Taiwanese (51.1%) was significantly higher than in aborigines, i.e., 6.4%, 3.9%, and 0% in Atayal, Paiwan, and Yami subjects, respectively. The percentage occurrence of ALDH-I deficiency and prevalence of alcohol dependence in Taiwanese and aborigines were negatively correlated. The predominant pattern of self-reported flush response after alcohol use among aborigines was of slow onset. The flush response to alcohol ingestion was examined in relation to aldehyde metabolizing enzyme. Since alcohol sensitivity is an important factor in the development and maintenance of the alcohol ingestion habit in humans, our results support the hypothesis that there is a biological basis in the different rates of alcohol abuse and dependence among different ethnic groups.

In the present study, the fumes generated from manual metal arc (MMA) and submerged metal arc (SMA) welding of low temperature service steel, and the chromium and nickel percentages in these fumes, were measured at various horizontal distances and vertical heights from the arc in order to obtain a three dimensional distribution. The MMA welding fume concentrations were significantly higher than the SMA welding fume concentrations. The highest fume concentration on the horizontal was shown in the fumes collected directly above the arc. The fume concentration vertically was highest at 50 cm height and reduced by half at 150 cm height. The fume concentration at 250 cm height was scarcely different from that at 150 cm height. The distribution of the chromium concentration vertically was analogous to the fume concentration, and a statistically significant difference in the chromium percentages was not found at the different heights. The nickel concentrations were not statistically significant within the welding processes, but the nickel percentages in the SMA welding fumes were statistically higher than in the MMA welding fumes. The highest nickel concentration on the horizontal was found in the fumes collected directly above the arc. The highest nickel concentration vertically showed in the fume samples collected at 50 cm height, but the greater the height the larger the nickel percentage in the fumes.

In the present study, the fumes generated from manual metal arc (MMA) and submerged metal arc (SMA) welding of low temperature service steel, and the chromium and nickel percentages in these fumes, were measured at various horizontal distances and vertical heights from the arc in order to obtain a three dimensional distribution. The MMA welding fume concentrations were significantly higher than the SMA welding fume concentrations. The highest fume concentration on the horizontal was shown in the fumes collected directly above the arc. The fume concentration vertically was highest at 50 cm height and reduced by half at 150 cm height. The fume concentration at 250 cm height was scarcely different from that at 150 cm height. The distribution of the chromium concentration vertically was analogous to the fume concentration, and a statistically significant difference in the chromium percentages was not found at the different heights. The nickel concentrations were not statistically significant within the welding processes, but the nickel percentages in the SMA welding fumes were statistically higher than in the MMA welding fumes. The highest nickel concentration on the horizontal was found in the fumes collected directly above the arc. The highest nickel concentration vertically showed in the fume samples collected at 50 cm height, but the greater the height the larger the nickel percentage in the fumes.

Vectorcardiogram (VCG) recorded by both the Frank and Kimura systems were examined in 45 patients with complete right bundle branch block (RBBB) and left axis deviation (LAD) to investigate the relationship seen on electrocardiogram (ECG) between RBBB with LAD and bilateral bundle. The sample included: 13 cases of type SI, SII, SIII, SaVF; 21 cases of type SI, SII, SIII, aVF; and 11 cases of types SI, SII, SIII. VCG recorded by the Frank system were classified into seven types according to the QRS loop pattern on the frontal plane and into three types according to the horizontal plane. The main findings were: (a) In the Frank system the QRS loop in the frontal plane showed a variety of patterns in RBBB with LAD. (b) On VCG of complete RBBB judged complicated by a left anterior hemiblock by the Frank system, the main portion of the QRS loop extended to the left superior or merely to the left in the frontal plane. The direction of rotation and position on the horizontal plane were not consistent. (c) The results of this study suggest the usefulness of the Kimura system as an auxiliary diagnostic technique.

Mesosomes of Staphylococcus aureus were examined morphologically under the electron microscope. Three different methods of specimen preparation (thin section, freeze-fracture, and negative staining) were used to eliminate artifacts due to sample processing. Mesosomes were rarely seen in intact cells but were quite distinct in autolysed cells incubated in 1.2M sucrose -0.33M acetate buffer (pH 5.8) at 25 degrees C. The phospholipid content increased by 20% of the control as autolysis proceeded. Since the plasma membrane did not show any other significant changes, the development of mesosomes during autolysis was shown to be a real event. Most of the well-developed mesosomes were found at the septum in negatively stained specimens. Initial wall-lysis occurred at this site, suggesting a close relationship between autolysis and the development of mesosomes in S. aureus.