In this study, pure strains that are capable of utilizing 2,4,6-trichlorophenol have been isolated from the mixed culture grown on substrates containing chlorophenolic compounds. Studies have been carried out on the capability of these isolated pure strains in suspended and immobilized forms to decompose 2,4,6-trichlorophenol. Additionally, the influence of primary substrates (e.g., phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol) on the decomposition of 2,4,6-trichlorophenol by the isolated pure strains grown in immobilized form is also investigated. The results are: Through bacterial isolation and identification, three pure strains have been obtained: Pseudomonas spp. strain 01, Pseudomonas spp. strain 02 and Agrobacterium spp. Whether in suspended or immobilized forms, all strains have poor removal efficiencies of 2,4,6-trichlorophenol. However, addition of 200 mg/l phenol will enable the immobilized Pseudomonas spp. strain 01, and Pseudomonas spp. strain 02 to achieve 65% and 48% removal of 2,4,6-trichlorophenol, respectively. Addition of phenol will assist the immobilized Pseudomonas spp. strain 02 in achieving removal of 2,4,6-trichlorophenol but the removal efficiency is not good if the phenol concentration is too low. The optimum phenol concentration should be between 200 and 400 mg/l.

Biodegradation, Environmental↗

PubMed2000

Removal of 2,4-dichlorophenol by suspended and immobilized Bacillus insolitus.

In this experiment, Bacillus insolitus was isolated and selected from a mixed culture that have been acclimated to chlorophenols. Decomposition of chlorophenolic compounds will be studied using this pure culture in both suspended and immobilized form. The results are: at lower initial concentrations of 2,4-dichlorophenol (10-50 mg/l), immobilized Bacillus insolitus shows a higher removal of 2,4-dichlorophenol than Bacillus insolitus in suspended growth. When the 2,4-dichlorophenol concentration becomes higher (50-200 mg/l), both immobilized and suspended Bacillus insolitus have approximately the same efficiency for removal of 2,4-dichlorophenol. Higher concentrations of 2,4-dichlorophenol are inhibitive to the growth of either suspended or immobilized Bacillus insolitus. At lower concentrations of 2,4-dichlorophenol, immobilized mixed culture may have the same removal efficiency of 2,4-dichlorophenol as immobilized pure culture of Bacillus insolitus. But with regard to the overall 2,4-dichlorophenol removal efficiency, immobilized pure culture is considered to be superior to immobilized mixed culture.

Bacillus↗

PubMed2000

Role of alcohol dehydrogenase E (ADHE) in the energy metabolism of Giardia lamblia.

Two hammerhead ribozymes flanked by Giardia lamblia alcohol dehydrogenase E (ADHE) antisense RNA fragments, ARzS and ARzL, were designed, synthesized and found capable of cleaving an ADHE mRNA fragment at the anticipated position in vitro. The ribozymes were then electroporated into Giardia trophozoites and expressed via the giardiavirus-mediated RNA expression system. Expression of the ribozyme with two short antisense arms, ARzS, was stabilized under puromycin selection and demonstrated a 33% reduction in ADHE mRNA and 25% decrease in NAD+-dependent ADH activity in the transfectants. Expression of ARzL, the ribozyme with two long antisense arms, cannot be enriched under puromycin without killing the transfected cells, probably due to excessive depletion of ADHE. Without the drug selection, however, transient expression of ARzL 20-40 h after electroporation resulted in an 83.7% loss of ADHE mRNA and an 84.5% reduction in ADH activity in the transfected cells. When the ribozyme moiety was removed from ARzL, the latter retained some of its in vivo activity of lowering ADHE mRNA and ADH activity, suggesting that inhibition of ADHE gene expression in Giardia can be accomplished by the antisense RNA alone, albeit less efficiently. The ADHE deficient transfectant demonstrated relatively poorer anaerobic growth but grew more vigorously than the wild type under aerobic conditions, suggesting that the role of ADHE in providing NAD+ through anaerobic reduction of acetyl-CoA to ethanol could be replaced by a yet unidentified aerobic enzyme(s) in Giardia. The close association consistently observed between the levels of ADHE mRNA and ADH activity in transfected Giardia cells suggests that ADHE could be the only functional alcohol dehydrogenase in Giaradia. One other Giardia gene encoding a putative Class III ADH, GIADH3, was identified and cloned, but no Class III ADH activity could be detected in Giardia by the conventional enzyme assays. This gene is thus probably unexpressed in Giardia trophozoite.

Aerobiosis↗

PubMed2000

The putative transcription initiation site in giardiavirus double-stranded RNA genome.

Explore the source record for details and available documents.

Animals↗

PubMed2000

Retentive and compressive strengths of modified zinc oxide-eugenol cements.

OBJECTIVES: This investigation sought to improve the handling and physical properties of a commonly used temporary zinc oxide-eugenol cement by changing the base/accelerator (B/A) ratio or combining it with a petroleum jelly or fluoride varnish. METHODS: Twelve modifications of a temporary cement were evaluated in terms of retentive strength, compressive strength at 24 h, film thickness and by scanning electron microscopy. RESULTS: Decreasing the B/A mixing ratio increased the retentive and compressive strengths, but reduced the film thickness of the cement. By increasing the percentage of incorporated petroleum jelly or fluoride varnish in the cement, there was a progressive decrease in the retentive and compressive strengths and in film thickness. CONCLUSIONS: Modifications of a zinc oxide-eugenol temporary cement to change the B/A ratio or to incorporate additives resulted in variations in physical properties. All modified forms of the cement had a film thickness less than 25 microns and a compressive strength below 35 MPa. With a wide range of retentive strength, modified forms of zinc oxide-eugenol cement may be found to have diverse clinical applications.

Analysis of Variance↗

PubMed2000

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells.

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.

Animals↗

PubMed2000

Repeated hypotensive episodes due to hepatic outflow obstruction during liver transplantation in adult patients.

We report two cases of unusual repeated hypotension, decreased cardiac output, decreased mixed venous oxygen saturation, decreased central venous pressure, pulmonary artery pressure, and pulmonary wedge pressure after the completion of all vascular anastamoses of liver transplantation. These unstable hemodynamics appear to reflect a clinically relevant picture of hypovolemia. However, the real cause was partial hepatic outflow obstruction. The obstruction was suspected because hypotension was alleviated by elevating the full-sized liver graft ventrally and to the left. Doppler ultrasound examination confirmed that the flow velocity of the hepatic vein outflow was insufficient when the liver fell to its resting position in the right hepatic fossa. An additional side-to-side cavo-caval anastomosis resolved the problem in one patient, whereas the other required not only the additional anastomosis, but also application of a tissue expander filled with 770 mL normal saline beneath the liver to eliminate the obstruction. We emphasize that obstruction of the hepatic outflow causes only temporal hypovolemia because of a decrease of venous return and that treatment of this complication should be surgical intervention to relieve the obstruction. Blind resuscitation with fluids will not solve the problem and, in fact, may result in fluid overload with subsequent complications.

Adult↗

PubMed2000

Inducible nitric oxide synthase inhibitors of Chinese herbs. Part 2: naturally occurring furanocoumarins.

Inducible nitric oxide synthase (iNOS)-dependent production of nitric oxide (NO) plays an important role in inflammation. The effects of various naturally occurring furanocoumarins on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells were evaluated in vitro. The results showed that angelicin, pimpinellin, sphondin, byakangelicol, oxypeucedanin, oxypeucedanin hydrate, xanthotoxin, and cnidilin are potential NO production inhibitors, and their IC50 values for inhibition of nitrite production were 19.5, 15.6, 9.8, 16.9, 16.8, 15.8, 16.6, and 17.7 microg/mL, respectively. Distinct structure-activity relationships were also revealed for the NO production inhibitory activities of these furanocoumarins. Activities of the angelicin type such as pimpinellin and sphondin were more potent than those of the psoralen type. Presence of a methoxy at the C6 position in the angelicin type seemed to be essential to augment the activity. Western blot analysis demonstrated that only sphondin dose-dependently inhibited the expression of the iNOS protein at 2.5-20 microg/mL. However, iNOS enzyme activity was stimulated with LPS for 12 h and sphondin was administered (20 microg/mL) for 24 h, which did not reasonably inhibit iNOS enzyme activity. L-NAME (100 microM), a known specific inhibitor of iNOS, was employed as a positive control with the same protocol and showed more than 50% inhibition activity. The results demonstrate that the NO production inhibitory activity of sphondin is due to the effect of iNOS expression, but not by direct inhibition of iNOS enzyme activity. Thus, sphondin may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.

Animals↗

PubMed2000

Nitric oxide synthase: models and mechanisms.

The overproduction or underproduction of nitric oxide has been implicated in pathological symptoms such as endotoxic shock, diabetes, allograft rejection, and myocardial ischimia/reperfusion injury. A thorough understanding of the biosynthesis of nitric oxide is necessary to probe and manipulate these signaling events. There is also considerable pharmacological interest in developing selective inhibitors of the several isoforms of nitric oxide synthase. The recently determined crystal structures of complexes between nitric oxide synthase and substrate, the mechanisms of the enzymatic reaction that generate nitric oxide and chemical precedents and models for these reactions are now coming into focus, but there are still numerous fascinating and unanswered questions regarding nitric oxide biosynthesis.

Animals↗

PubMed2000

International sharing of split liver grafts in Asia: initial experience.

The donor shortage problem is particularly serious in Asia and has markedly limited progress in liver transplantation. The increasing demand has, in fact, made it necessary to resort to living donor liver transplantation in both pediatric and adult recipients. Nevertheless, expanding the use of split liver allografts is yet another option to increase the supply. This has a wide potential application on a regional level because most liver transplant programs are still small and may have limited resources in terms of being able to do two transplants in one sitting. The first experience of overseas sharing of split liver grafts in Asia took place in January 1999. The graft was from a 35-yr-old donor from Kaohsiung, Taiwan, who sustained irreversible brain damage in a vehicular accident and had optimal conditions for multiorgan donation. The liver was split ex vivo and the left lateral segment was given to a 3-yr-old girl with biliary atresia at the Chang Gung Memorial Hospital. The extended right lobe split graft was transported to Hong Kong and transplanted into a 51-yr-old male patient with end-stage hepatitis C cirrhosis who was then in a state of acute failure with hepatorenal syndrome. Graft function was excellent in both recipients and the patient from Taiwan was discharged without any complications. Unfortunately, the Hong Kong recipient developed a cerebrovascular accident and required a reoperation for bile leakage from the cut surface of the liver in the early postoperative period. He has made a steady recovery since then; graft function has remained good and his kidneys have recovered. Both patients are currently alive and well 11 months post-transplant. This initial experience of overseas sharing of split liver grafts in Asia demonstrates its feasibility. It has a potentially wide applicability and could lead to the establishment of a formal organ-sharing network in the region. Established competence and mutual trust among the participating liver transplant teams would be essential in perpetuating such a graft-multiplying strategy on an organized basis.

Adult↗

PubMed2000

P2Y(2) receptor-mediated proliferation of C(6) glioma cells via activation of Ras/Raf/MEK/MAPK pathway.

1. Extracellular purine and pyrimidine nucleotides have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C(6) glioma cells responsible for stimulating cell proliferation associated with mitogen-activated protein kinase (MAPK) activation. 2. UTP and ATP produced a similar effect on [(3)H]-thymidine incorporation in a time- and concentration-dependent manner, suggesting the involvement of P2Y(2) receptor in mediating proliferation of C(6) glioma cells. 3. In response to UTP, both p42 and p44 MAPK were activated in a time- and concentration-dependent manner using Western blot analysis with an anti-phospho-p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5 min and declining by 30 min. 4. Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. 5. UTP-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP. 6. These results conclude that the mitogenic effect of UTP mediated through a P2Y(2) receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in cultured C(6) glioma cells.

Adenosine Triphosphate↗

PubMed2000

Tumour necrosis factor-alpha- and interleukin-1beta-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells.

The elevated levels of inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF-alpha and IL-1beta on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. TNF-alpha and IL-1beta induced [(3)H]-thymidine incorporation in a time- and concentration-dependent manner. The maximal stimulation of [(3)H]-thymidine incorporation induced by TNF-alpha and IL-1beta was seen 12 h after incubation with these cytokines. In response to TNF-alpha and IL-1beta, p42/p44 MAPK was activated with a concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF-alpha and IL-1beta. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)-phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)-PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. TNF-alpha- and IL-1beta-induced [(3)H]-thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF-alpha and IL-1beta were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF-alpha- and IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in TSMCs.

Animals↗

PubMed2000

Lipopolysaccharide enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.

Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen-activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)-induced inositol phosphates (IPs) accumulation and Ca(2+) mobilization in canine cultured tracheal smooth muscle cells (TSMCs). LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentration-dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achieved with 100 microg ml(-1) LPS. Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation and Ca(2+) mobilization. However, there was no effect on the IPs response induced by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF-BB enhanced BK-induced IPs response. These enhancements by LPS and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)) in TSMCs, characterized by competitive inhibition of [(3)H]-BK binding using B(1) and B(2) receptor-selective reagents. The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Animals↗