Biomedical subjects
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[formula: see text] Lewis and/or Bronsted acid additives permit ring opening and halogenation of spiroketals at substantially reduced temperatures to produce omega-iodo enol ethers in improved yield and purity, which can undergo further reaction in the presence of distal electrophilic centers to give new steroid skeletons.
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OBJECTIVE: To evaluate the clinical applications of the detection of the stable end products of NO, nitrite and nitrate (NO2-./NO3-.) in sputum from patients with asthma. METHODS: 45 patients with asthma at remission (group I, n = 15) or exacerbation state with mild attack (group II, n = 15) or moderate to severe attack (group III, n = 15), 15 cases of chronic cor pulmonale secondary to chronic bronchitis at exacerbation (group IV), and 20, normal control subjects (group V n = 20) were included in this study. NO2-./NO3-. in sputum and serum were measured by Griess reaction, while the eosinophil cationic protein (ECP) in sputum and pulmonary function were also measured in tested patients with asthma. RESULTS: The median concentrations of sputum NO2-./NO3-. in group I, II and III were 85, 131 and 136 mumol/L respectively, all were significantly higher than that in controls (63 mumol/L); those of group II and III were significantly higher than that of group I, while that of group IV (33 mumol/L) was significantly lower than those of the asthmatic groups and the control. The level of NO derivatives in sputum was closely correlated with the degree of airflow obstruction and sputum ECP levels in patients with asthma at remission (r = -0.587, P < 0.05; r = 0.535, P < 0.01 respectively) and exacerbation (r = -0.485, P < 0.05; r = 0.563, P < 0.05 respectively). There was no significant correlation between serum and sputum NO2-./NO3-., and no significant correlation between serum NO2-./NO3-. and the degree of airflow obstruction. A significant decrease in the concentration of sputum NO2-./NO3-. was observed after therapeutic treatment of patients with moderate to severe attack (136 vs 93 mumol/L). CONCLUSION: The level of NO2-./NO3-. in sputum may be a useful marker for monitoring the state of airway inflammation and assessing the therapeutic efficacy.
OBJECTIVE: To explore the effects of the antibodies to LCAMs on proliferation of pulmonary fibroblast (L929 cell line). METHODS: Proliferation of fibroblasto and level of collagen production were determined by (3)H-TDR incorporation and ELISA. RESULTS: The supernate of AMs cultured with anti-LCAMs could inhibit the proliferation of fibroblast. The presence of anti-CD(11)B (125 microg/ml) could significantly inhibit the proliferation of fibroblast (P < 0.05), and reduce collagen secretion by 29.7%. When anti-CD(18) and anti-CD(54) at the concentration of 25 microg/ml, 5 microg/ml respectively, the inhibition rate of fibroblast proliferation differed from that in control group (P < 0.05). moreover combination of the two antibodies resulted in more significant inhibition effect than that seen by the two antibodies separately. CONCLUSIONS: The increased expression of CAMs on AM might play an important role in the pathogenesis of the pulmonary fibrosis and MAbs to CD(11)B/CD(18), CD(54) possess a potential regiment for intervention of pulmonary fibrosis.
OBJECTIVE: To assess the tissue oxygenation status and the effects of mechanical ventilation (MV) on oxygen dynamics in COPD patient with hypercapnia. METHODS: Gastric intramucosal pH (pHi) measured by gastric tonometer was examined in 19 patients before and after MV. RESULTS: pHi calculated before MV was 7.18 +/- 0.06; After receiving MV, pHi increased gradually until above 7.32 in 4 days; during weaning, there were no significant changes in pHi before and after discontinuation. The correlation analysis showed that there was no correlation between pHi and pHa (r = 0.352, P > 0.05), PgCO(2) and PaCO(2) (r = 0.421, P > 0.05), suggesting that pHi measurement would not be affected by hypercapnia. CONCLUSIONS: Our study showed that tissue oxygenation insufficiency existed in COPD exacerbation stage. Gastric pHi may be used as the parameter to estimate tissue oxygenation status and a predictor of weaning from mechanical ventilation.
OBJECTIVE: Using anti-transforming growth factor-beta(1) antibody binding to transforming growth factor-beta(1) (TGF-beta(1)) in conditioned supernatant obtained from alveolar macrophages (AM) in broncho-alveolar lavage fluid (BALF) in bleomycin-induced rats, we investigated the effect of binding of TGF beta(1) to anti-TGF beta(1) antibody on proliferation and synthesis of collagen by fibroblast in pulmonary fibrosis. METHODS: Bleomycin (0.75 mg/100 BW) was instilled intratracheally into wistar rats, then broncho-alveolar lavage (BAL) was performed 7 days later. AMs (5 x 10(5)/ml) were cultured in 0.2% FCS RPMI-1640 medium in vitro for 24 hours and conditioned medium was obtained. L929 fibroblast (7 x 10(3)/well) was cultured in 96-well microtiter plate with conditioned medium and in different doses of anti- TGF beta(1) antibody (1, 5, 10, 20 microg/ml) and IgG for 24 hours respectively. (3)H-Tdr was added 1 microCi per well for 6 hours before culture finished. The proliferation of fibroblast was studied by (3)H-Tdr incorporation rate and synthesis of collagen type IV was tested by ELISA. RESULTS: (1) Fibroblast proliferation which was induced by AM conditioned medium could be significantly depressed by anti-TGF beta(1) antibody in vitro (P < 0.01), and it appeared in a dose-dependent manner. (2) Synthesis of collagen type IV was depressed about 32% by 10 microg/ml anti-TGF beta(1) antibody. CONCLUSIONS: Fibroblast proliferation and collagen synthesis could be depressed by anti-TGF beta(1) antibody in vitro. It seems possible to provide a new way for the treatment of pulmonary fibrosis.
OBJECTIVE: To compare subclinical infection rate in the vaccinated group with type I inactivated vaccine against hemorrhagic fever with renal syndrome (HFRS) with that in the controls and to understand its enhancement. METHODS: A trial field was selected in Jiande County, Zhejiang Province during July 1974 to November 1997. Paired-sera before and after vaccination were collected from 401 vaccinee and 360 controls, respectively. Serum titer of indirect immunofluorescent IgG antibody (IFAT) against HFRS was detected for each of them, and its cut-off value depended on the distribution of serum antibody titer in the second determination in the controls, which could be used to evaluate subclinical infection before vaccination. RESULTS: There was no significant difference in subclinical infection between those with positive and negative IFAT before vaccination, with different cut-off values for identifying subclinical infection. In both vaccinated and control groups with negative IFA before vaccination, subclinical infection rate was 7.62% in the controls, and 2.17% and 1.63% in the vaccinated ones, with cut-off values of 1:160 and 1:320, respectively, significantly different from that in the former. Subclinical infection rate was 11.38% and 6.78% in the vaccinated ones, as cut-off values of 1:20 and 1:40, respectively, without significant difference from the controls. CONCLUSION: No increase in subclinical infection in the vaccinated group with type I inactivated vaccine against HFRS was found.
Contents of magnolol and honokiol in 76 samples of Magnolia officinalis collected from 11 counties in Zhejiang, Fujian, Sichuan, Guangxi, Hunan, Jiangxi and Hubei are analyzed by means of HPLC. A study on the effect of tree age on effective ingredients in Magnolia offcinalis shows that the influence of tree age on the content of magnolol and honokiol is correlated with the varieties under cultivation, but a small effect on the content of the phenols with an indent at the tip of leaves. The content of magnolol in other types of leaves increases rapidly with increase in age, diameter of the trunk and thickness of the bark, with not many changes found at the age of 12 or up. Increase in age may be favorable to the full expression of oily characteristic. These results provide a scientific base for the determination of optimal time for harvesting bark from artificially established Magnolia officinalis stands.
Dichloroacetic acid (DCA), a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clinically in the management of lactic acidosis. DCA is biotransformed to glyoxylic acid by glutathione-dependent cytosolic enzymes in vitro and is metabolized to glyoxylic acid in vivo. The enzymes that catalyse the oxygenation of DCA to glyoxylic acid have not, however, been identified or characterized. In the present investigation, an enzyme that catalyses the glutathione-dependent oxygenation of DCA was purified to homogeneity (587-fold) from rat liver cytosol. SDS/PAGE and HPLC gel-filtration chromatography showed that the purified enzyme had a molecular mass of 27-28 kDa. Sequence analysis showed that the N-terminus of the purified protein was blocked. An internal sequence of 30 amino acid residues was obtained that matched the recently discovered human glutathione transferase Zeta well [Board, Baker, Chelvanayagam and Jermiin (1997) Biochem. J. 328, 929-935]. Western-blot analysis showed that the purified rat-liver enzyme cross-reacted with rabbit antiserum raised against recombinant human glutathione transferase Zeta. The apparent Km and Vmax values of the purified enzyme with DCA as the variable substrate were 71.4 microM and 1334 nmol/min per mg of protein, respectively; the Km for glutathione was 59 microM. Both the purified rat-liver enzyme and the recombinant human enzyme showed high activity with DCA as the substrate. These results demonstrate that the glutathione-dependent oxygenation of DCA to glyoxylic acid is catalysed by a Zeta-class glutathione transferase.
Dichloroacetic acid (DCA) is a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clinically in the management of lactic acidosis. Recent studies show that glutathione transferase Zeta (GSTZ) catalyzes the oxygenation of DCA to glyoxylic acid [Tong et al. (1998) Biochem. J. 331, 371-374]. In the present studies, the substrate selectivity of GSTZ, the kinetics of DCA metabolism, and the fate of DCA and glutathione were investigated. The results showed that GSTZ catalyzed the oxygenation of bromochloro-, bromofluoro-, chlorofluoro-, dibromo-, and dichloroacetic acid, but not difluoroacetic acid, to glyoxylic acid. GSTZ also catalyzed the biotransformation of fluoroacetic acid to S-(carboxymethyl)glutathione, and of (R,S)-2-bromopropionic acid, (R)-, (S)-, and (R,S)-2-chloropropionic acid, and (R, S)-2-iodopropionic acid, but not (R,S)-2-fluoropropionic acid, to S-(alpha-methylcarboxymethyl)glutathione; and of 2, 2-dichloropropionic acid to pyruvate. No biotransformation of 3, 3-dichloropropionic acid was detected, and no GSTZ-catalyzed fluoride release from ethyl fluoroacetate and fluoroacetamide was observed. The relative rates of DCA biotransformation by hepatic cytosol were mouse > rat > human. Immunoblotting showed the presence of GSTZ in mouse, rat, and human liver cytosol. 13C NMR spectroscopic studies showed that [2-13C]glyoxylic acid was the only observable, stable metabolite of [2-13C]DCA. Also, glutathione was required, but was neither consumed nor oxidized to glutathione disulfide, during the oxygenation of DCA to glyoxylic acid. These results are consistent with a reaction mechanism that involves displacement of chloride from DCA by glutathione to afford S-(alpha-chlorocarboxymethyl)glutathione, which may undergo hydrolysis to give the hemithioacetal S-(alpha-hydroxycarboxymethyl)glutathione. Elimination of glutathione from the hemithioacetal would give glyoxylic acid.
Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2-5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378-2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2-5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.
Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.
In the aquatic environment, diet is an important route of exposure for the common contaminant and procarcinogen benzo(a)pyrene (BaP). Dietary organisms vary in their BaP content and in contaminated areas often contain other xenobiotics including cytochrome P4501A inducers. This study examined the effect of dose and previous dietary exposure to the inducer ss-naphthoflavone (BNF) upon the intestinal metabolism of BaP and the systemic bioavailability of BaP-derived products in catfish. BaP was administered at 2 and 20 microM into in situ-isolated perfused intestines of control and BNF-pretreated catfish. The intestine formed an array of metabolites in all treatments including potentially hazardous metabolites such as BaP-7,8 and 9,10 dihydrodiols and 6-methyl-BaP. BNF treatment disproportionally increased the contribution of BaP-7,8 and 9,10 dihydrodiols relative to the contributions of other metabolites. A greater percentage of metabolites was evident as conjugates in 2 microM controls, whereas a greater percentage of unconjugated metabolites was evident for 20 microM controls and BNF treatments of both dosages. BNF pretreatment and the higher 20 microM BaP dosage resulted in greater bioavailability, with 2.6-5.5-fold and 3.0-6. 3-fold increases in systemically available BaP products, respectively. Metabolites represented 10.2-23.1% of the increased bioavailability with BNF treatment, suggesting that mechanisms, in addition to induced metabolism, may be operative. These results indicate that intestinal bioavailability, level of biotransformation, and the metabolic profile of BaP-derived products entering the blood from the intestine may be altered by dose and dietary BNF pretreatment.
This study evaluates the transcriptional and post-transcriptional regulation of prolactin (PRL) by vasoactive intestinal peptide (VIP). Pituitary nuclei from laying (control), incubating (with enhanced VIP secretion), and VIP-immunized laying turkey hens, and from pituitary cells cultured with or without VIP were used in nuclear run-on transcription assays. Cytoplasmic PRL mRNA was analyzed by slot blot hybridization. PRL transcription was greater in hyperprolactinemic incubating birds (PRL/beta-actin=3.33) than in laying birds (PRL/beta-actin=1.83). VIP-immunoneutralized birds had 47% and 51% decreases in PRL transcription and cytoplasmic PRL mRNA, respectively when compared with laying birds. In primary pituitary cell cultures, VIP significantly increased the transcription rate of PRL (3.8-fold) and cytoplasmic PRL mRNA (3.2-fold) compared with that of non-VIP-treated pituitary cells. The stability of pre-existing PRL mRNA was measured by Northern blot analysis after addition of actinomycin D. PRL mRNA half-lives were calculated using a two-component model, with a first-long component of 18.0+/-1.0 h and a second-short component of 3.7+/-0.7 h in non-VIP-treated pituitary cells. Both half-lives were significantly increased (53. 2+/-6.9 and 26.3+/-4.3 h) in VIP-treated cells. The present data show that VIP acts to stimulate PRL expression by up-regulating the transcription rate of PRL and by enhancing PRL mRNA stability.
To characterize further vasoactive intestinal peptide (VIP) as the prolactin-releasing factor in avian species, the present study examined hypothalamic VIP transcription and plasma prolactin (PRL) levels during the turkey reproductive cycle. The contribution of transcription to hypothalamic VIP mRNA steady-state levels and VIP content in response to gonadal stimulating photoperiod was also investigated. Nuclear run-on transcription assays were performed using nuclei isolated from hypothalami. Cytoplasmic VIP mRNA levels, and VIP content in the median eminence and plasma PRL levels were determined by Northern blot analysis and radioimmunoassays respectively. The alterations in VIP transcription mirrored the changes in cytoplasmic VIP mRNA and VIP content during the reproductive stages. VIP transcription, cytoplasmic VIP mRNA level and VIP content were lowest in non-photostimulated birds, higher (P<0.05) in laying hens, and greatest (P<0.05) in incubating birds. These increases paralleled the changes in circulating plasma PRL levels. Changes in VIP transcription (P>0.05) were not observed during the transition from incubation to photorefractoriness, even though there was a sharp decline in circulating plasma PRL levels (P<0.05). Following photostimulation, VIP transcription, cytoplasmic VIP mRNA levels, and VIP content increased as the hens progressed towards sexual maturity (P<0.05), and these increases were correlated with an increased plasma PRL level. These results suggest that VIP is regulated in large part at the transcriptional level during the turkey reproductive cycle and that this transcriptional regulation is coupled to the photo-induced increase in PRL secretion.
An analysis of effective ingredients in 20 to 29 year old Magnolia officinalis of three different leaf types coming from various sources shows that the content of phenols differ extremely significantly in various leaf types which are a key to determination of quality of Magnolia officinalis also varies significantly among different sources which only have a significant effect on the content of magnolol. Varieties and types cultivated in different places are responsible for the variation in content of magnolol. The conclusions obtained from this study coincide with the traditional insight into quality of Magnolia officinalis.
Human subjects were exposed by inhalation to 250, 500, and 1000 ppm 1,1-dichloro-1-fluoroethane (HCFC-141b) for 4 hr, and urine samples were collected from 0-4, 4-12, and 12-24 hr for metabolite analysis. 19F nuclear magnetic resonance spectroscopic analysis of urine samples from exposed subjects showed that 2,2-dichloro-2-fluoroethyl glucuronide and dichlorofluoroacetic acid were the major and minor metabolites, respectively, of HCFC-141b. Urinary 2, 2-dichloro-2-fluoroethyl glucuronide was hydrolyzed to 2, 2-dichloro-2-fluoroethanol by incubation with beta-glucuronidase, and the released 2,2-dichloro-2-fluoroethanol was quantified by gas chromatography/mass spectrometry. Concentrations of 2, 2-dichloro-2-fluoroethanol were highest in the urine samples collected 4-12 hr after exposure, but 2,2-dichloro-2-fluoroethanol was also detected in the samples collected 0-4 and 12-24 hr after exposure. Exposure concentration-dependent excretion of 2, 2-dichloro-2-fluoroethanol, obtained by hydrolysis of 2, 2-dichloro-2-fluoroethyl glucuronide, was observed in seven of the eight subjects studied. In conclusion, HCFC-141b is metabolized in human subjects to 2,2-dichloro-2-fluoroethanol, which is conjugated with glucuronic acid and excreted as its glucuronide in urine in a time- and exposure concentration-dependent manner.
This study hypothesized that depressive experiences of the elderly could be aggravated by universal factors such as low social status, poor health, financial strain, and unhealthy lifestyle, as well as by factors specific to an indigenous socio-cultural environment (stressful family dynamics) of a given population. Three hundred and fifty Chinese subjects aged 65 or older were interviewed either at their homes or in the geriatric out-patient clinic of Beijing Hospital. Hierarchical logistic regression was used to examine significant predictors of depression. Results showed that certain social status, poor physical health, financial strain, unhealthy lifestyle, and stressful family situation explained 47 percent of the variance in depression. However, stressful family situation alone explained 13 percent of the variance in depression, indicating that family factors were important predictors of depression for Chinese elderly. Furthermore, this study demonstrated for the first time that verbal abuse within Chinese families is a significant correlate of depression among the elderly. Cultural implications of these findings are discussed.