Genomic organization of a cellulase gene family in Phanerochaete chrysosporium

Summary Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.     

Protective effects of calmodulin antagonists (trifluoperazine and W-7) on hypothermic ischemic rat hearts

The cardioprotective effect of calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) was examined on the isolated rat heart exposed to hypothermic and ischemic conditions by measuring distribution of lysosomal enzymes in myocardial cells, and leakage of creatine kinase (CK) during reperfusion and postischemic recovery in myocardial systolic function. Experimental hearts were infused with 20 degrees C Krebs-Henseleit bicarbonate buffer (KHB) or KHB containing TFP or W-7 for 2min every 30min during hypothermic ischemia. After ischemia for 120min at 20 degrees C, rat hearts were reperfused at 37 degrees C for 30min. TFP and W-7 improved functional recovery and prevented CK release. In TFP treated hearts, leakage of lysosomal enzymes was reduced significantly, whereas stabilization of lysosomes by W-7 did not occur. These results suggest that calcium-calmodulin dependent enzymes may play an important role in the development of cellular damage of the myocardium during hypothermic ischemia, although levels of leakage of lysosomal enzymes may be unreliable predictors of functional recovery after hypothermic ischemia.     

Homologous expression of Phanerochaete chrysosporium manganese peroxidase,using bialaphos resistance as a dominant selectable marker

Manganese peroxidase (MnP) is a major extracellular component of the lignin-degrading system of the white-rot fungus, Phanerochaete chrysosporium. Homologous expression of recombinant MnP isozyme 1 (rMnP1) in P. chrysosporium was achieved using a novel transformation system for this fungus, which utilizes the Streptomyces hygroscopicus bialaphos-resistant gene, bar, as the selectable marker. The transformation frequency for this system is approximately 100 bialaphos-resistant transformants per microgram of plasmid DNA. Transformed strains all contain plasmid DNA, ectopically integrated into the fungal genome. Using this transformation system, the promoter region of the P. chrysosporium translation elongation factor gene was used to drive expression of mnp1, encoding MnP1, in primary metabolic cultures of P. chrysosporium, where endogenous MnP was not expressed. Approximately 2–3 mg of active recombinant MnP1 per liter of extracellular medium was produced in agitated cultures of transformants.Communicated by U. Kück     

The nature of extra-chromosomal maintenance of transforming plasmids in the filamentous basidiomycete Phanerochaete chrysosporium

Summary The nature of extra-chromosomal maintenance of the transforming plasmid p12-6 in Phanerochaete chrysosporium was studied. Our results indicate that the plasmid is maintained in the fungal transformants extra-chromosomally as part of a larger endogenous plasmid (designated pME) of P. chrysosporium. Using the total DNA of p12-6 fungal transformants, p12-6, as well as a larger plasmid, p511, were recovered in recA ^– E. coli strains while only p12-6 was recovered in recA ⁺ E. coli strains. The results also showed that the cytosine methylation system has no apparent effect on the strain-dependent recovery of p12-6 and p511 in E. coli from the total DNA of fungal transformants.     

Effects of calmodulin antagonist (W-7) on phorbol ester (PMA)-induced contractile response in isolated rat aorta.

The aim of this study was to investigate effects of calmodulin antagonist (W-7) on the contractile response of the rat aorta induced by activation of protein kinase C (PKC) by phorbol ester. Phorbol 12-myristate 13-acetate (PMA) produced biphasic contraction i.e., a sustained contraction (initial contraction) and 17.9 +/- 1.7 min later, this progressively developed contraction was changed to a delayed contraction superimposed on the initial contraction. The delayed contraction was completely inhibited by treatment with nicardipine. The onset of the delayed contraction was significantly delayed by treatment with W-7, whereas same concentration of W-7 showed a weak relaxant effect (10%) on the PMA-induced maximal contraction of aorta. Higher concentration of W-7 strongly inhibited PMA-induced sustained contraction. These results suggest that PMA-induced biphasic contractile response may be regulated by calmodulin.     

Effect of protein kinase C inhibitor (H-7) and calmodulin antagonist (W-7) on pertussis toxin-induced IL-1 production by human adherent monocytes. Comparison with lipopolysaccharide as a stimulator of IL-1 production.

Human adherent monocytes stimulated with 1 microgram/ml pertussis toxin (PT) produced interleukin-1 (IL-1), as measured by thymocyte co-stimulation assay and enzyme-linked immunosorbent assay (ELISA), specific for IL-1 alpha and IL-1 beta. To clarify the role of protein kinase C (PKC) and calmodulin in IL-1 production, we investigated the effects of a PKC inhibitor, H-7, and a calmodulin antagonist, W-7 on PT- and lipopolysaccharide (LPS)-induced IL-1 production by monocytes. Addition of 10 microM and 20 microM H-7 to the culture medium markedly suppressed both PT- and LPS-induced IL-1 production. PT-induced IL-1 production was significantly suppressed by 5 microM and 10 microM W-7. However, LPS-induced IL-1 production was not suppressed by W-7 at the concentrations tested. When monocytes were labelled with Quin 2/AM, IL-1 production by monocytes stimulated with PT and LPS was markedly suppressed. These results indicate that different pathways are involved in the IL-1 production by PT and LPS; both calmodulin- and PKC-dependent processes are necessary for the IL-1 production induced by PT, whereas LPS-induced IL-1 production is dependent on the PKC. Inhibition of IL-1 production by interfering with intracellular Ca2+ trafficking in Quin 2/AM-loaded monocytes may be associated with the inhibition of PKC and calmodulin activity.     

Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in Saccharomyces cerevisiae

A cDNA fragment encoding the Phanerochaete chrysosporium cellobiohydrolase (cbh1-4) was amplified and cloned with the aid of the polymerase chain reaction (PCR) technique. The cbh1-4 gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase (end1) gene were successfully expressed in Saccharomyces cerevisiae under the control of the phosphoglycerate kinase-I (PGK1) and alcohol dehydrogenase-II (ADH2) gene promoters and terminators, respectively. The native P. chrysosporium signal sequence mediated secretion of cellobiohydrolase in S. cerevisiae, whereas secretion of the endo-β-1,4-glucanase was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1 _S ). These constructs, designated CBH1 and END1, respectively, were expressed separately and jointly in S. cerevisiae. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of CBH1 and END1 synergistically enhanced cellulose degradation by S. cerevisiae. Received: 1 March 1996 / 9 April 1996     

Relationship of chemical structures of textile dyes on the pre-adaptation medium and the potentialities of their biodegradation by Phanerochaete chrysosporium

Azo dye derivatives of azobenzene constitute the largest group of dyes used in the textile industry and possess recalcitrant chemical groups, such as those of azo and sulphonic acid. Some microorganisms are able to degrade these aromatic compounds. In the present work, decolourisation of culture media containing azo dyes by the ligninolytic fungus Phanerochaete chrysosporium was achieved under nitrogen-limited conditions. The dyes used in the study are derivatives of meta- or para-aminosulphonic or aminobenzoic acids and include in their structures groups such as guaiacol or syringol, which are bioaccessible to the lignin degrading fungus P. chrysosporium. The aim of this study was to pre-adapt the microorganism to the structure of the dyes and to establish the relationships of the chemical structure of the dye present in the pre-adaptation medium with the chemical structure of the dye to be degraded. The azo dye used in the pre-adaptation medium that gave the best overall decolourisation performance was a meta-aminosulphonic acid and guaiacol derivative. The azo dye derivative of a meta-aminobenzoic acid and syringol showed a better performance in the decolourisation assays. Preliminary GC-MS studies indicated the formation of a nitroso substituted catechol metabolite, a precursor of aromatic ring cleavage, which was confirmed to occur by an enzymatic assay. The presence of this type of metabolite allows the establishment of a possible metabolic pathway towards mineralisation.     

The effect of a converting enzyme inhibitor on autoregulation and intrarenal distribution of glomerular filtration in the rat

The role of the renin-angiotensin system in the autoregulation and distribution of the single nephron glomerular filtration rate (SNGFR) in anaesthetized, normotensive rats was investigated. SNGFR in outer cortical (OC) and inner cortical (IC) nephrons of the left kidney were measured with a modified Hanssen technique at three levels of renal arterial pressure (RAP): at a spontaneous arterial pressure; at a value within the autoregulatory limit, 100 mmHg; and at the lower limit of the autoregulatory range, 70 mmHg. This was done in control rats and in rats given a continuous i.v. infusion of the converting enzyme inhibitor (CEI) captopril (3 mg . h-1 X kg-1 BW). In control rats there was complete autoregulation of SNGFR in both OC and IC nephrons when RAP was reduced to 100 mmHg. Further reduction to 70 mmHg elicited different responses among the cortical layers, associated with a decrease in SNGFR. A fractional redistribution of glomerular filtration rate towards IC nephrons was evident. Administration of CEI at spontaneous RAP increased SNGFR in IC nephrons compared with values in control rats, but did not notably alter SNGFR in OC nephrons. Reduction of RAP to 100 mmHg during CEI infusion caused SNGFR to decrease below control values in both OC and IC nephrons, and the autoregulation as found in control rats was impaired. When RAP was lowered to 70 mmHg during CEI administration there was a progressive decrease in SNGFR in all cortical layers, although absolute changes were much greater in IC nephrons than in OC nephrons.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of a protein kinase C inhibitor, H-7, on human neutrophil oxidative burst and degranulation

The role of C-kinase in the activation of human polymorphonuclear leukocytes has been examined using H-7, a recently described C-kinase inhibitor. We found that H-7 will inhibit PMN superoxide anion release in response to the tumor promotor phorbol myristate acetate and the calcium ionophore A23187. In contrast, no inhibition by H-7 was seen for PMN superoxide release stimulated by the chemotactic peptide FMLP. H-7 did not inhibit PMN NADPH oxidase activity or PMN degranulation by any stimulant, but it reversed a phorbol ester-induced inhibition of granule release by FMLP. The results show that H-7 differentially affects the PMN functional events of secretion and superoxide release and suggests that an H-7 inhibitable C-kinase is not involved in chemotactic peptide induced activation of PMN and may not regulate stimulus induced PMN degranulation.     

Trifluoperazine, a calmodulin inhibitor, blocks secretion in cultured chromaffin cells at a step distal from calcium entry

Trifluoperazine, a calmodulin antagonist, inhibited the secretory response of cultured bovine adrenal medullary chromaffin cells to acetylcholine (10(-4) M) or a depolarizing concentration of [K+] (56 mM KCl) in a dose-related fashion. The ID50s of this effect were 2 x 10(-7) M and 2.2 x 10(-6) M for acetylcholine and high [K+], respectively. A decrease in external [Ca2+] concentration of the incubation medium from 4.4 to 0.275 mM resulted in an increase in the percentage of inhibition produced by trifluoperazine on the acetylcholine-evoked secretory response from 20.7 to 96.5%, respectively. However, trifluoperazine inhibited the acetylcholine-evoked catecholamine output by a similar absolute magnitude for all [Ca2+] concentrations tested with the exception of 4.4 mM [Ca2+]. Trifluoperazine, unlike the [Ca2+] channel blocker Ni2+, in concentrations (10(-6)-10(-5) M) that were found to inhibit significantly [K+]-induced amine output did not modify [K+]-induced 45Ca uptake or 45Ca efflux. However, trifluoperazine at a concentration of 2.5 x 10(-5) M was found to produce a small decrease in the 45Ca efflux curve and a decrease in the [K+]-evoked 45Ca uptake of 30 +/- 14% (n = 6). In addition, 2.5 x 10(-6) M trifluoperazine, a concentration which was found to suppress high [K+]-induced amine release by 64 +/- 5%, did not inhibit the 45Ca2+-Ca2+ exchange mechanism. These results demonstrate that trifluoperazine, an antipsychotic agent with anticalmodulin activity, blocks catecholamine release from cultured chromaffin cells at a step distal from calcium entry and, consequently, suggests a role for calmodulin in the secretory process of these cells.     

Spinal effects of the calmodulin inhibitor calmidazolium on dorsal horn neurons in the rat

Drugs able to inhibit calmodulin activation can prevent some consequences of the rise in intracellular calcium. It has recently been shown that intrathecal injection of calmodulin inhibitors induce analgesia in rats. We study here the effect induced by the calmodulin inhibitor, calmidazolium, on the activity of dorsal horn neurons driven by noxious and non-noxious stimuli. Extracellular recordings of convergent (n = 12), low-threshold mechanoreceptive (n = 5) and proprioceptive (n = 5) units were made in the presence of calmidazolium. Calmidazolium (600 micrograms) reduced the noxious (50 degrees C) heat-evoked responses obtained in convergent neurons. On the contrary, the non-noxious tactile responses obtained in low-threshold mechanoreceptive neurons as well as the joint movement-evoked responses obtained in proprioceptive units remained unmodified. We conclude that calmidazolium can block nociceptive processing in the spinal cord and that this fact can help to explain the analgesic effects that intrathecal W-7 and calmidazolium induce in behavioral tests.     

Effects of a calmodulin inhibitor on bleomycin-induced lung inflammation in hamsters. Biochemical, morphometric, and bronchoalveolar lavage data.

Previous studies have shown that bleomycin-induced pulmonary fibrosis is accompanied by elevated levels of calcium and calmodulin, which are important in the regulation of many biologic processes. The authors have further extended these observations and assessed the effect of a calmodulin inhibitor, trifluoperazine, on bleomycin-induced lung damage with biochemical, morphometric, and bronchoalveolar lavage techniques. The cumulative mortality due to bleomycin was not significantly reduced in animals receiving trifluoperazine. Trifluoperazine had no apparent effect on lung levels of collagen and DNA elevated by bleomycin. However, morphometric studies showed that the volume density of the lesion, the volume of amorphous material and interstitial inflammation, and the number of monocytes within lesions were less in the lungs of bleomycin-treated hamsters receiving trifluoperazine daily. When compared with hamsters treated with bleomycin alone, animals treated with both bleomycin and trifluoperazine had significantly fewer lymphocytes in their bronchoalveolar lavage fluid. The data suggest that trifluoperazine reduced the acute inflammation which accompanies bleomycin pneumotoxicity but did not affect the subsequent development of pulmonary fibrosis. It has been postulated that the observed antiinflammatory action of trifluoperazine may be due to inhibition of calmodulin-dependent leukocyte functions.     

血管紧张素转换酶抑制剂对损伤后动脉弹性蛋白酶的影响

目的：研究球囊导管损伤后早期血管紧张素转换酶抑制剂对动脉中膜弹性蛋白酶的影响。方法：12周雄性Wistar大鼠颈动脉和主动脉用球囊导管损伤,分成实验组和对照组,分别于术前2 d投与血管紧张素转换酶抑制剂（temocapril-HCl, 10 mg·kg^-1·d^-1）和溶剂,术后2、3、5和10 d处死。用原位杂交、免疫组织化学和酶活性测定研究弹性蛋白酶。结果：实验组动脉损伤10 d时的内膜面积与对照组相比明显被抑制（P<0.01）,实验组2、3和5 d弹性蛋白酶mRNA阳性细胞率、弹性蛋白酶阳性细胞率及其活性明显低于对照组（P<0.01, P<0.05）。结论：ACEI（temocapril-HCl）明显抑制损伤后动脉中膜平滑肌弹性蛋白酶表达和活性,提示弹性蛋白酶的表达与血管紧张素转换酶的作用有关。