Identification of Yersinia enterocolitica using a random genomic DNA microarray chip.

AIMS: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. METHODS AND RESULTS: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. CONCLUSIONS: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. SIGNIFICANCE AND IMPACT OF THE STUDY: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.

Controlled release of an antibiotic, gentamicin sulphate, from gravity spun polycaprolactone fibers.

The antibiotic, gentamicin sulphate (GS), was incorporated in gravity-spun polycaprolactone (PCL) fibers by spinning from particulate suspensions of the drug in PCL solution to produce a controlled delivery system. The production rate of GS-loaded PCL fibers was confined to the range 1-1.5 m/min and the fiber diameter to 170-220 microm. The kinetics of drug release could be adjusted by varying the GS loading of the fibers and the suspension preparation conditions. Gradual release of approximately 80% of the initial GS content was measured in phosphate buffered saline at 37 degrees C over 50 days from fibers spun from nonhomogenized suspensions, whereas loss of this amount of antibiotic occurred in less than 10 days from fibers spun from homogenized suspensions. Studies of growth inhibition of Stapyhlococcus epidermidis in culture indicated that GS released after 2 weeks from PCL fibers retained antibacterial activity. This behavior recommends further investigation of PCL fibers for local delivery of antibiotics to combat infection associated with periodontal disease, musculoskeletal injuries, and implantation of fiber-based tissue substitutes such as vascular prostheses.

Production of mycelia and exo-biopolymer from molasses by Cordyceps sinensis 16 in submerged culture.

The molecular weight of exo-biopolymer obtained from a submerged culture of Cordyceps sinensis 16 consisted of a main unit and a subunit of 126 and 68 kDa, respectively. The optimal medium for the production of mycelia and exo-biopolymer was determined to be molasses containing 2% sucrose, 0.9% yeast extract, 0.3% K2HPO4, and 0.4% CaCl2. Using optimized medium, maximum productions of mycelia and exo-biopolymer in shake-flask culture were 54.0 g/L and 28.4 g/L, respectively. This study suggests that large-scale production of mycelia and exo-biopolymer by C. sinensis 16 is possible in submerged culture.

Delivery of the antibiotic gentamicin sulphate from precipitation cast matrices of polycaprolactone.

Microporous, poly(epsilon-caprolactone) (PCL) matrices were loaded with the aminoglycoside antibiotic, gentamicin sulphate (GS) using the precipitation casting technique by suspension of powder in the PCL solution prior to casting. Improvements in drug loading from 1.8% to 6.7% w/w and distribution in the matrices were obtained by pre-cooling the suspension to 4 degrees C. Gradual release of approximately 80% of the GS content occurred over 11 weeks in PBS at 37 degrees C and low amounts of antibiotic were measured up to 20 weeks. The kinetics of release could be described effectively by the Higuchi model with the diffusion rate constant (D) increasing from of 1.7 to 5.1 microg/mg matrix/day(0.5) as the drug loading increased from 1.4% to 8.3% w/w. GS-loaded PCL matrices retained anti-bacterial activity after immersion in PBS at 37 degrees C over 14 days as demonstrated by inhibition of growth of S. epidermidis in culture. These findings recommend further investigation of precipitation-cast PCL matrices for delivery of hydrophilic molecules such as anti-bacterial agents from implanted, inserted or topical devices.

Precipitation casting of drug-loaded microporous PCL matrices: incorporation of progesterone by co-dissolution.

Microporous, poly(epsilon-caprolactone) (PCL) matrices were loaded with progesterone by precipitation casting using co-solutions of PCL and progesterone in acetone. Progesterone loadings up to 32% w/w were readily achieved by increasing the drug content of the starting PCL solution. The kinetics of steroid release in PBS at 37 degrees C over 10 days could be described effectively by a diffusional release model although the Korsmeyer-Peppas model indicated the involvement of multiple release phenomena. The diffusion rate constant (D) increased from 8 to 24 microg/mg matrix/day0.5 as the drug loading increased from 3.6 to 12.4% w/w. A total cumulative release of 75%-95% indicates the high efficiency of steroid delivery. Increasing the matrix density from 0.22 to 0.39 g/cm3, by increasing the starting PCL solution concentration, was less effective in changing drug release kinetics. Retention of anti-proliferative activity of released steroid was confirmed using cultures of breast cancer epithelial (MCF-7) cells. Progesterone released from PCL matrices into PBS at 37 degrees C over 14 days retarded the growth of MCF-7 cells by a factor of at least 3.5 compared with progesterone-free controls. These findings recommend further investigation of precipitation-cast PCL matrices for delivery of bioactive molecules such as anti-proliferative agents from implanted, inserted or topical devices.

Differences in p53 gene polymorphisms between Korean schizophrenia and lung cancer patients.

The reduced incidence of cancer observed in schizophrenia patients may be related to differences in genetic background. It has been suggested that genetic predisposition towards schizophrenia is associated with reduced vulnerability to lung cancer, and p53 gene is one of the candidate genes. We tested the genetic association between schizophrenia and lung cancer by analyzing polymorphic sites in the p53 gene. Genotype and allele frequencies at two polymorphic sites in the p53 gene (BstUI and MspI restriction sites in exon 4 and intron 6, respectively) were studied in Korean schizophrenia (n=179) and lung cancer patients (n=104). Comparisons of the genotype and allele frequencies of the MspI polymorphism revealed significant differences between schizophrenia and lung cancer patients. The results suggest that the p53 polymorphism specifically found in schizophrenia patients may be associated with reduced vulnerability to lung cancer.

Analysis of alkali-soluble glucan produced by Saccharomyces cerevisiae wild-type and mutants.

The alkali-soluble glucan of the yeast cell wall contains beta-(1,3)- and (1,6)-D-linkages and systemically enhances the immune system. To isolate Saccharomyces cerevisiae mutants producing glucan with a high degree of beta-(1,6)-D-glycosidic bonds, a wild-type strain was mutagenized with ultraviolet light. The mutants were then selected by treatment with 1.0 mg laminarinase, endo-beta-(1,3)-D-glucanase/ml. The alkali-soluble glucan was extracted by modified alkalysis followed by the Cetavlon method and concanavalin-A chromatography. The prepared alkali-soluble glucans from the wild-type and the mutants were compared with respect to yield and polymer structure using gas chromatography, 13C-NMR spectrometry, high performance liquid, and multi-angle laser light scattering and refractive index detectors. The results indicated that the S. cerevisiae mutants had ten-fold more alkali-soluble glucan than the wild-type. Structural analysis revealed that the alkali-soluble glucan from the mutants also had a higher degree of beta-(1,6)-D-linkage than that from the wild-type.

In vitro model using mouse hepatocytes for study of alcohol stress.

In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5 to approximately 5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.

Effects of polysaccharides from rhizomes of Curcuma zedoaria on macrophage functions.

The effects of Curcuma zedoaria, which is used as a condiment, in perfumery, and as a medicine, on immune response were investigated by measuring macrophage-stimulating activity in macrophages and RAW 264.7 cells. In this study, CZ-1 and CZ-1-III, the fractions partially purified from C. zedoaria, had a strong, dose-dependent lysosomal enzyme activity. It was suggested that active portions of CZ-1-III were polysaccharides rather than proteins. Phagocytic activity increased as a similar pattern in both the gram-negative and gram-positive bacteria, time-dependently. It was demonstrated that CZ-1-III can augment the oxygen burst response but had an even higher activity in vivo than in vitro. Also a significant increase of H2O2, NO, and TNF-alpha production was observed. However, the production of TNF-alpha at the concentration of 1,000 microg/ml decreased. These data suggested that C. zedoaria had macrophage-stimulating activity and the possibility of being used as a biological response modifier.

Alteration of the NAD+/NADH ratio in CHO cells by stable transfection with human cytosolic glycerol-3-phosphate dehydrogenase: resistance to oxidative stress.

The intracellular level of the NAD+/NADH ratio plays a vital role in sustaining and coordinating the catabolic reaction of the cell, and reflects the redox state of cytosol. Antioxidants play a role to protect cytosol and membrane from free radicals. This role of antioxidants involves sustaining cell viability and the procedure is thought to be regulated by the equilibrium of the redox state of the cell. However, there is very little known about how the NAD+/NADH level is set and changed. To alter the ratio, human NAD-dependent glycerol-3-phosphate dehydrogenase (cGPDH) cDNA was transfected stably in CHO dhfr- cells. When compared to parental CHO cells, cGPDH activities of the transfected cells were increased 8-12 fold, but the NAD+/NADH ratio was decreased. Specific growth rate of the transfected cells was similar to or slight lower than that of wild type CHO cells. Cell viability of the stable transformants against H2O2 was increased without change of either catalase or glutathione peroxidase activity. However, the increase of cell viability was correlated with the decrease of NAD+/NADH ratio in transfectants. From these results, it is suggested that the overexpression of cGPDH changes the NAD+/NADH ratio toward a decrease, and by this change in the redox state the cell confers more resistance against H2O2.

Body position, membrane diffusing capacity and pulmonary capillary blood volume in chronic bronchitis and pulmonary emphysema.

BACKGROUND: The effect of body position on diffusing capacity and its components, membrane diffusing capacity (Dm) and pulmonary capillary blood volume (Vc), in patients with chronic obstructive pulmonary disease (COPD) has remained elusive. This study was designed to evaluate the effect of body position on diffusing capacity for carbon monoxide (DLco), Dm and Vc in male patients with chronic bronchitis and pulmonary emphysema. METHODS: Pulmonary function tests including spirometry and lung volume were assessed in the erect position, and DLco, Dm and Vc were measured in the erect and supine positions in a random order in 17 men with chronic bronchitis and 19 men with pulmonary emphysema. RESULTS: Spirometry results and lung volumes were comparable between both groups of patients; however, significantly lower values of DLco and Kco (DLco corrected by alveolar volume, VA) were observed in the emphysema than in the bronchitis group. In the bronchitis group, Kco and Vc were significantly higher in the supine than in the erect position, but Dm was significantly lower in the supine position. Alternation of body position did not significantly affect DLco and its components in the emphysema group. DLco, Kco and Vc in both the erect and supine positions were significantly higher in the bronchitis than in the emphysema group. Vc-SE (SE, the data in the supine minus those in the erect position) was also significantly higher in the bronchitis group. In the bronchitis group, DLco-SE was significantly correlated with Dm-SE and Vc-SE. However, Kco-SE was highly correlated with Dm-SE. In the emphysema group, DLco-SE and Kco-SE were highly correlated with Vc-SE only. CONCLUSIONS: An increase in Vc in the supine position may account for the postural effect on Kco in bronchitis patients. In patients with pulmonary emphysema, decreased DLco and an absence of postural effect on DLco and its components may be due to a widespread abnormality of the pulmonary capillary bed. These findings may be of value in elucidating the difference in mechanisms of impaired gas exchange between patients with chronic bronchitis and pulmonary emphysema.

Changes of nuclear glycoproteins in normal and simian virus 40-transformed WI-38 human lung fibroblasts.

To investigate changes in nuclear glycoproteins after transformation, nuclei and nuclear extracts were prepared from the normal and SV40-transformed WI-38 human lung fibroblasts grown in vitro. The nuclei of the normal and transformed cells were isolated by a vigorous pipetting method, and the isolated nuclei were verified by morphological and biochemical examinations. The nuclear glycocomponents, which were labelled with fluorescein isothiocyanate-lectins, were evenly found all through the isolated nuclei. Lectin blotting analysis showed that dozens of nuclear proteins contain both Man and GlcNAc moieties. The glycoproteins bearing the terminal N-acetylglucosamine sugar moieties were also demonstrated by in vitro galactosylation with [3H]-galactose. The result showed that nine glycoproteins specifically appeared or disappeared upon viral transformation or as SV40 replication products, indicating that there are significant differences in nuclear glycoproteins between the normal and SV40-transformed cells. These results suggest that the modification of Man and GlcNAc moieties are dynamic changes.

Therapeutic effects of diuretics and paracentesis on lung function in patients with non-alcoholic cirrhosis and tense ascites.

BACKGROUND/AIMS: Ascites may cause or aggravate pulmonary dysfunction in patients with liver cirrhosis. Diuretics and paracentesis are the main therapies for ascites. The aim of the present study was to evaluate and compare the therapeutic effects of diuretics and large-volume paracentesis on lung function in 26 male patients with non-alcoholic cirrhosis and tense ascites. METHODS: The patients were divided into two groups. Group A was composed of 13 subjects who were treated with diuretics including spironolactone (100-400 mg/day) and furosemide (80-320 mg/day). In group B, 13 subjects received large-volume paracentesis plus intravenous albumin (6-8 g/l ascites removed). Pulmonary function tests including spirometry, plethysmography, single-breath carbon-monoxide diffusing capacity (DLco) and arterial blood gases, were done 1 day before diuretic treatment and 1 day after termination of the study in group A patients, and 1 day before and after large-volume paracentesis in group B subjects. RESULTS: Before treatment, the clinical and laboratory data were comparable between the two groups. After treatment, ventilatory function as evidenced by forced expiratory volume in 1 s, forced vital capacity, total lung capacity, functional residual capacity and expiratory reserve volume, and DLco increased significantly in both groups. Arterial PO2 and PCO2 increased significantly and AaPO2 (alveolar-arterial PO2 difference) decreased significantly in the subjects treated with diuretics. Nevertheless, paracentesis did not improve arterial blood gases. The changes in lung volumes, DLco and PaO2 after treatment (the data after minus those before treatment) were comparable, except that a significant decrease in AaPO2 was observed in the diuretic group. CONCLUSIONS: Both diuretic therapy and large-volume paracentesis significantly improved the ventilatory function in patients with tense cirrhotic ascites. In terms of oxygenation improvement as evaluated by AaPO2, diuretic treatment may be superior to large-volume paracentesis.

Effects of ascites and body position on gas exchange in patients with cirrhosis.

The effects of ascites and body position on gas exchange were evaluated in 22 male cirrhotics. All underwent spirometry and plethysmography in the sitting (erect) position. Single breath carbon monoxide diffusing capacity (DLco) was measured in the erect and supine positions in a random sequence. A significantly negative correlation was found between Kco (DLco corrected by alveolar volume [VA]) and lung volumes. DLco was significantly higher in the erect than in the supine position. On the other hand, Kco was significantly higher in the supine position. The differences of DLco and Kco between the erect and supine positions (the data in the supine minus those in the erect position) correlated highly with FEV1 and FVC. Thirteen patients underwent large volume paracentesis, and pulmonary function tests were done one day before and after paracentesis. Lung volumes and DLco increased significantly after paracentesis. In contrast, Kco decreased significantly. A significantly negative correlation was found between the change of Kco before and after paracentesis and that of lung volumes (the data after minus those before paracentesis). There was no remarkable change in the differences of DLco and Kco between the two positions after paracentesis. It is concluded that ascites may prevent further worsening of gas exchange and attenuate the postural effect on gas exchange in cirrhotic patients with significant amounts of ascites.

Effect of large-volume paracentesis on pulmonary function in patients with cirrhosis and tense ascites.

The effect of large-volume paracentesis on lung function was evaluated in 12 male patients with cirrhosis. All underwent pulmonary function tests including spirometry, plethysmography and single-breath carbon-monoxide diffusing capacity 1 day before and after paracentesis. The amount of ascitic fluid removed ranged from 3.6 to 131 (mean +/- SD, 7.4 +/- 3.01). After paracentesis, forced vital capacity, forced expiratory volume at 1 s, total lung capacity, functional residual capacity, inspiratory capacity, expiratory reserve volume, diffusing capacity and alveolar volume increased significantly. In contrast, Kco (diffusing capacity corrected by alveolar volume) decreased significantly. After paracentesis, the increase in diffusing capacity was highly correlated with lung volumes and the amount of removed ascitic fluid. Nevertheless, a significantly negative correlation was found between the change of Kco before and after paracentesis and that of lung volumes. The increase in lung volumes and ventilation to the lower lungs with unfavorable ventilation-perfusion matching might explain the discrepancy between changes in diffusing capacity and Kco after large-volume paracentesis. In conclusion, these results suggest that pulmonary function in patients with cirrhosis and tense ascites is partly improved by large-volume paracentesis. Large-volume paracentesis might be useful for symptomatic relief in selected patients with tense ascites.

Effect of body position on gas exchange in patients with unilateral central airway lesions. Down with the good lung?

In this study, we evaluated the effect of body position (erect, supine, and two decubitus positions) on gas exchange (alveolar-arterial PO2 difference [AaPO2]) in 35 patients who had various degrees of lung collapse roentgenographically caused by unilateral central airway lesions, with special reference to the difference in AaPO2 between two lateral decubitus positions. The patients were divided into two groups. Group 1 was composed of 23 patients with FEV1/FVC > 70 percent. In group 2, there were 12 patients with FEV1/FVC < 70 percent. Our results showed that the mean AaPO2 of group 1 patients was least in the lateral decubitus position with normal lung down (AaPO2N), followed by those in the supine position (AaPO2S), in the lateral decubitus position with lesioned lung down (AaPO2L), and in the erect position (AaPO2E). There was no significant difference in AaPO2 obtained in four positions. However, a significantly negative correlation was found between AaPO2NL (AaPO2N minus AaPO2L) and patient's FEV1 (p < 0.05). In group 2 patients, the mean AaPO2E was least, followed by AaPO2L, AaPO2N, and AaPO2S. The changes of body position did not significantly affect gas exchange in group 2 patients. Unlike previous reports, the present study showed that AaPO2N was not exclusively less than AaPO2L in our patients. AaPO2N was higher than AaPO2L in 11 of 23 in group 1 and in 5 of 12 in group 2 patients. In summary, our results indicated that positional changes did not significantly affect gas exchange in the patients with lung collapse roentgenographically caused by unilateral central airway lesions and the dogma "Down with the good lung" could not be applied to these patients flawlessly.

Effects of body position and age on membrane diffusing capacity and pulmonary capillary blood volume.

The effects of body position and age on the membrane diffusing capacity (Dm), pulmonary capillary blood volume (Vc), and the single breath carbon monoxide diffusing capacity (Dco) were evaluated in the erect (sitting) and supine positions in 16 normal young men (under 40 years old, younger group) and in 13 older men (over 40 years old, older group). Dm and Vc were estimated by several measurements of the Dco at increasing alveolar oxygen tension (PAO2). The results showed that Dco, Dm, Vc, and Kco (Dco corrected by alveolar volume) decreased with age in both positions. The differences in Dco, VC, and Kco between the two positions (supine minus the erect position) also decreased with age. The mechanisms of the increases in Dco in the supine position remain to be explained but may be due to a change in pulmonary capillary shape from an elliptical (erect position) to a circular configuration (supine position) since Vc increased more than Dm on assuming the supine position. The findings may be of clinical importance since many physicians have attempted to utilize a reduction in the positional change in Dco as a potential marker of disease.

Regulation and intracellular localization of the biotin holocarboxylase synthetase of 3T3-L1 cells.

A quantitative assay has been developed to measure holocarboxylase synthetase activity in cellular extracts. This assay was based on measuring the incorporation of [3H]biotin of high specific activity (4.3 Ci/mmol) into purified rat liver apopyruvate carboxylase. With this assay, holocarboxylase synthetase in 3T3-L1 mouse fibroblasts has been monitored. During the differentiation of this cell from a fibroblast to an adipocyte, holocarboxylase synthetase activity was found to increase threefold, while pyruvate carboxylase activity rose 20-fold. The results suggest a possible relationship between the activity of the holocarboxylase synthetase and the level of the biotin-dependent carboxylases within the mammalian cell. Utilizing digitonin fractionation. the intracellular distribution of this enzyme has also been examined. In the 3T3-L1 cell, the large majority (approximately 70%) of the total holocarboxylase synthetase activity was found in the cytosolic compartment.